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Papers by rex pratt

Research paper thumbnail of β-Lactamase Inhibitors: Non-β-Lactams

Research paper thumbnail of The Perfect Penicillin? Inhibition of a Bacterial DD-Peptidase by Peptidoglycan-Mimetic β-Lactams

Journal of the American Chemical Society, 2004

Streptomyces R61 was a generous gift from Professor J.-M. Frère (University of Liège, Liège, Belg... more Streptomyces R61 was a generous gift from Professor J.-M. Frère (University of Liège, Liège, Belgium). Lα-aminopimelic acid was synthesized as described by Rosowsky et al. S1 The substrate 1 was prepared by Dr. John W. Anderson in this laboratory S2 ; the synthesis of 4 will be described in a separate publication. All other reagents were from commercial sources and used as received. 1 H NMR † spectra were obtained from a Varian Gemini-300 MHz spectrometer. IR spectra were recorded on a Perkin Elmer 1600 FTIR instrument. Preparative thin layer chromatography (TLC) was performed on Analtech 20x20 cm, 2000micron F254 plates. The plates were visualized with UV light. Electrospray mass spectra were obtained from the

Research paper thumbnail of Synthesis and Evaluation of New Substrate Analogues of Streptomyces R61 dd -Peptidase:  Dissection of a Specific Ligand

The Journal of Organic Chemistry, 2004

Good substrates of the Streptomyces R61 dd-peptidase, such as glycyl-L-alpha-amino-epsilon-pimely... more Good substrates of the Streptomyces R61 dd-peptidase, such as glycyl-L-alpha-amino-epsilon-pimelyl-D-alanyl-D-alanine, 1 (Anderson, J. W.; Pratt, R. F. Biochemistry 2000, 39, 12200-12209), contain the glycyl-L-alpha-amino-epsilon-pimelyl side chain. A number of thia variants of this structure have been synthesized by means of a disconnection strategy whereby the appropriate thiols were reacted with either acryloyl-D-alanyl-D-alanine or haloalkanoyl-D-alanyl-D-alanines. Kinetics studies of the hydrolysis of these compounds by the R61 DD-peptidase showed that the presence of the N-terminal glycylammonium ion and the pimelyl-alpha-carboxylate are very important for efficient catalysis. The results of deletion of the C-terminal D-alanine indicate a promising direction toward new inhibitors. Shorter (one methylene less) and longer (one methylene more) analogues of 1 are also poor substrates. Molecular modeling and dynamics studies suggest that the higher mobility of the active site residues and the modified substrates in enzyme-substrate complexes may be the dominant factor in this loss of reactivity. The general conclusion is that essentially all of the structural elements of the side chain of 1 are required to produce a good substrate. This result has important implications for the design of inhibitors of DD-peptidases.

Research paper thumbnail of Crystal Structure of the Bacillus subtilis Penicillin-binding Protein 4a, and its Complex with a Peptidoglycan Mimetic Peptide

Journal of Molecular Biology, 2007

The genome of Bacillus subtilis encodes 16 penicillin-binding proteins (PBPs) involved in the syn... more The genome of Bacillus subtilis encodes 16 penicillin-binding proteins (PBPs) involved in the synthesis and/or remodelling of the peptidoglycan during the complex life cycle of this sporulating Gram-positive rod-shaped bacterium. PBP4a (encoded by the dacC gene) is a low-molecular mass PBP clearly exhibiting in vitro DD-carboxypeptidase activity. We have solved the crystal structure of this protein alone and in complex with a peptide (D-αaminopymelyl-ε-D-alanyl-D-alanine) that mimics the C-terminal end of the Bacillus peptidoglycan stem peptide. PBP4a is composed of three domains: the penicillin-binding domain with a fold similar to the class A β-lactamase structure and two domains inserted between the conserved motifs 1 and 2 characteristic of the penicillin-recognizing enzymes. The soaking of PBP4a in a solution of D-α-aminopymelyl-ε-D-alanyl-D-alanine resulted in an adduct between PBP4a and a D-α-aminopimelyl-ε-D-alanine dipeptide and an unbound D-alanine, i.e. the products of acylation of PBP4a by D-α-aminopymelyl-ε-D-alanyl-D-alanine with the release of a D-alanine. The adduct also reveals a binding pocket specific to the diaminopimelic acid, the third residue of the peptidoglycan stem pentapeptide of B. subtilis. This pocket is specific for this class of PBPs.

Research paper thumbnail of Structures of Two Kinetic Intermediates Reveal Species Specificity of Penicillin-binding Proteins

Journal of Molecular Biology, 2002

Research paper thumbnail of Crystal Structures of Complexes of Bacterial dd-Peptidases with Peptidoglycan-Mimetic Ligands: The Substrate Specificity Puzzle

Journal of Molecular Biology, 2008

The X-ray crystal structures of covalent complexes of the Actinomadura R39 DD-peptidase and Esche... more The X-ray crystal structures of covalent complexes of the Actinomadura R39 DD-peptidase and Escherichia coli penicillin-binding protein (PBP) 5 with βlactams bearing peptidoglycan-mimetic side chains have been determined. The structure of the hydrolysis product of an analogous peptide bound noncovalently to the former enzyme has also been obtained. The R39 DDpeptidase structures reveal the presence of a specific binding site for the D-αaminopimelyl side chain, characteristic of the stem peptide of Actinomadura R39. This binding site features a hydrophobic cleft for the pimelyl methylene groups and strong hydrogen bonding to the polar terminus. Both of these active site elements are provided by amino acid side chains from two separate domains of the protein. In contrast, no clear electron density corresponding to the terminus of the peptidoglycan-mimetic side chains is present when these β-lactams are covalently bound to PBP5. There is, therefore, no indication of a specific side-chain binding site in this enzyme. These results are in agreement with those from kinetics studies published earlier and support the general prediction made at the time of a direct correlation between kinetics and structural evidence. The essential highmolecular-mass PBPs have demonstrated, to date, no specific reactivity with peptidoglycan-mimetic peptide substrates and β-lactam inhibitors and, thus, probably do not possess a specific substrate-binding site of the type demonstrated here with the R39 DD-peptidase. This striking deficiency may represent a sophisticated defense mechanism against low-molecular-mass substrate-analogue inhibitors/antibiotics; its discovery should focus new inhibitor design.

Research paper thumbnail of Formation and Structure of 1:1 Complexes between Aryl Hydroxamic Acids and Vanadate at Neutral pH

Inorganic Chemistry

Although aryl hydroxamic acids are well-known to form coordination complexes with vanadate (V V),... more Although aryl hydroxamic acids are well-known to form coordination complexes with vanadate (V V), the nature of these complexes at neutral pH and submillimolar concentrations, the conditions under which such complexes inhibit various serine amidohydrolases, is not well established. A series of qualitative and quantitative experiments, involving UV/vis, 1 H NMR, and 51 V NMR spectroscopies, established that both 1:1 and 1:2 vanadate/hydroxamate complexes form at pH 7.5, with the former dominating at submillimolar concentrations. Formation constants for the complexes of several aryl and alkyl hydroxamic acids were determined; for example, for benzohydroxamic acid, the stepwise formation constants of the 1:1 and 1:2 complexes were 3000 and 400 M-1 , respectively. The 51 V chemical shift of the 1:1 4-nitrobenzohydroxamic acid complex was −497 ppm, and that of its unsubstituted analogue was −498 ppm. A 1 H− 15 N HSQC spectrum of the 4-nitrobenzo-15 N-hydroxamic acid/vanadate complex indicated the presence of an N−H group with 15 N and 1 H chemical shifts of 115 and 5.83 ppm, respectively. A 13 C NMR spectrum of the complex of 4-nitrobenzo-13 C-hydroxamic acid with vanadate displayed a resonance at 170.1 ppm and thus a coordination-induced shift (CIS) of +3.8 ppm. In contrast, the CIS value of an established 1:2 complex, thought to contain chelated hydroxamic acid ligands, was +11.9 ppm. These spectral data led to the following structural picture of 1:1 complexes of vanadate and aryl hydroxamic acids. They contain penta-or hexa-coordinated vanadium. The ligand is in the hydroxamate rather than hydroximate form. The ligand is presumably bound to vanadium through the hydroxamic hydroxyl oxygen, but the hydroxamic acid carbonyl oxygen interacts weakly with vanadium. These species are the most likely candidates for the inhibitors of serine amidohydrolases found in vanadate/ hydroxamic acid mixtures.

Research paper thumbnail of Benzopyranones with retro-amide side chains as (inhibitory) β-lactamase substrates

Bioorganic & Medicinal Chemistry Letters, 2004

Benzopyran derivatives R 0350 Benzopyranones with Retro-Amide Side Chains as (Inhibitory) β-Lacta... more Benzopyran derivatives R 0350 Benzopyranones with Retro-Amide Side Chains as (Inhibitory) β-Lactamase Substrates.-3-Carbamoylbenzopyranones (I) are synthesized (no yields are given) and found to be comparably effective as substrates of typical class A, C and D β-lactamases as analogous benzopyranones bearing 'normal' amide side chains. The new 8-carboxy derivative (Ib), however, forms a much more tightly-bound acyl-enzyme with a class C β-lactamase compared to its 'normal' analogue, and thus provides a structural lead to new inhibitors of this class of β-lactamase.-(ADEDIRAN, S. A.; CABARET, D.;

Research paper thumbnail of Synthesis and β-lactamase reactivity of α-substituted phenaceturates

Bioorganic & Medicinal Chemistry, 2006

Lactams with 6a (penicillins) or 7a (cephalosporins) substituents are often b-lactamase inhibitor... more Lactams with 6a (penicillins) or 7a (cephalosporins) substituents are often b-lactamase inhibitors. This paper assesses the effect of such substituents on acyclic b-lactamase substrates. Thus, a series of m-carboxyphenyl phenaceturates, substituted at the glycyl a-carbon by-OMe,-CH 2 OH,-CO 2 À , and-CH 2 NH 3 + , have been prepared, and tested for their reactivity against serine b-lactamases. The latter two are novel substituents in b-lactamase substrates. The methoxy and hydroxymethyl compounds were found to be poor to moderately good substrates, depending on the enzyme. The aminomethyl compound gave rise to a transiently stable (t 1/2 = 4.6 s) complex on its reaction with a class C b-lactamase. The reactivity of the compounds against three low molecular weight DD DD-peptidases was also tested. Again, the methoxy and hydroxymethyl compounds proved to be quite good substrates with no sign of inhibitory complexes. The DD DD-peptidases reacted with one enantiomer (the compounds were prepared as racemates), presumably the D D compound. The class C b-lactamase reacted with both D D and L L enantiomers although it preferred the latter. The structural bases of these stereo-preferences were explored by reference to the crystal structure of the enzyme by molecular modeling studies. The aminomethyl compound was unreactive with the DD DDpeptidases, whereas the carboxy compound did not react with any of the above-mentioned enzymes. The inhibitory effects of the-OMe and-CH 2 OH substituents in b-lactams apparently require a combination of the substituent and the pendant leaving group of the b-lactam at the acyl-enzyme stage.

Research paper thumbnail of Inhibition of Class A and C β-Lactamases by Diaroyl Phosphates

Biochemistry, 2009

A series of diaroyl phosphates was employed to assess the general reactivity of this class of mol... more A series of diaroyl phosphates was employed to assess the general reactivity of this class of molecule against classical class A and class C beta-lactamases. The compounds were found, in general, to be inhibitory substrates of both classes of enzyme. In each case, they reacted rapidly with the enzyme (10(4) to 10(6) s(-1) M(-1)) to yield transiently stable intermediates, most likely acyl-enzymes, which slowly (10(-3) to 10(-1) s(-1)) regenerated free enzyme. In certain cases, side branches from direct turnover produced EII complexes ("substrate" inhibition), more inert EI' complexes, and, in one case, a completely inactive EI' complex. Deacylation, but not acylation, was enhanced by electron-withdrawing substituents. Acylation rates were enhanced by hydrophobic substitution, both in the diaroyl phosphate and at the enzyme active site. The latter factor led to the general order of beta-lactamase acylation rates: class D (previous results) > class C > class A. It is likely that nanomolar inhibitors of all serine beta-lactamases could be achieved by rational exploitation of diacyl phosphates.

Research paper thumbnail of Intramolecular Cooperativity in the Reaction of Diacyl Phosphates with Serine β-Lactamases

Biochemistry, 2009

Asymmetric diaroyl phosphates (ArCOOPO 2 − OCOAr′, where Ar = Ph, Ar′ = 4-biphenyl, 2benzothiophe... more Asymmetric diaroyl phosphates (ArCOOPO 2 − OCOAr′, where Ar = Ph, Ar′ = 4-biphenyl, 2benzothiophenyl and 2-benzofuranyl), have been prepared, evaluated as serine (classes A, C, D) βlactamase inhibitors, and compared with respect to the latter with their symmetric "parents", where Ar = Ar′. The asymmetric compounds, in general, were found to react with the β-lactamases in two modes, corresponding to different orientations with respect to the active site, whereby either of the two aroyl groups may acylate the enzyme to form two different inert acyl-enzymes, E-COAr and E-COAr′. In all cases, the asymmetric compounds, in one orientation, react more rapidly with the enzymes tested than one symmetrical "parent" but not both. From comparisons of activation free energy differences, it was found that the changes in free energy on changing from one aryl group to another, in either the acyl group or the leaving group, were not additive, i.e. that the effect of changing one aroyl group to another depended on the leaving group and vice versa. Thus, intramolecular cooperativity between the aroyl groups must exist, arising either from direct interaction between them or from protein-mediated interaction, or from a combination of both. Such cooperativity brings fresh opportunities and challenges to the search for novel ß-lactamase inhibitors. Aroyl phosphates, 1, have proven to be very effective inhibitors of serine ß-lactamases (1-4). The phosphate leaving group appears to interact with polar resides in a way as to enhance active site acylation by both specific amidoacyl groups and non-classical aroyl groups (2-4,5,6). Acylation by the latter leads to hydrolytically refractive acyl-enzymes and thus to significant inhibition (Scheme 1). A series of diaroyl phosphates 2 have proven to be particularly effective inhibitors (3,4). Structure-activity studies have shown that hydrophobic substituents entrance inhibition, largely through enhancement of acylation rates, while electron-donating substituents enhance inhibition by depression of deacylation rates (3).

Research paper thumbnail of Mechanism of Reaction of Acyl Phosph(on)ates with the β-Lactamase of Enterobacter cloacae P99

Biochemistry

A series of acyl phosph(on)ates has been prepared to more closely examine the details of the inte... more A series of acyl phosph(on)ates has been prepared to more closely examine the details of the interactions of this class of molecule with beta-lactamases. In general, they were found to react with the class C beta-lactamase of Enterobacter cloacae P99 in two ways, by acylation and by phosphylation. The acyl-enzymes generated by the former reaction were transiently stable with half-lives of between 3 and 45 s, of comparable lifetime therefore to those generated by the inhibitory beta-lactams cefotaxime, cefuroxime, and cefoxitin. On the other hand, phosphylation led to a completely inactive enzyme. In general, the second-order rate constants for acylation (k(cat)/K(m)) were larger than for phosphylation (k(i)). As expected on chemical grounds, phosphylation was found to be relatively more effective for the phosphonates than the phosphates. The acyl phosphates were much more effective acylating agents however. The acylation reaction was found to be enhanced by hydrophobic substituents in both the acyl and leaving group moieties. Thus, the most reactive compound in this series was benzo[b]thiophene-2-carbonyl 2'-naphthyl phosphate with a K(m) value of 0.15 microM and a k(cat) of 0.2 s(-1); k(cat)/K(m) is therefore 1.3 x 10(6) s(-1) M(-1), making this compound the most specific acyclic acylation reagent for this beta-lactamase yet described. Significant substrate inhibition by this compound suggested that further binding regions may be available for exploitation in inhibitor design. A linear free energy analysis showed that the transition states for acylation of the beta-lactamase by aroyl phosphates are analogues of the corresponding aryl boronic acid adducts. Molecular modeling suggested that the aroyl phosphates react with the P99 beta-lactamase with the aroyl group in the side chain/acyl group site of normal substrates and the phosphate in the leaving group site. In this orientation, the phosphate leaving group interacts strongly with Lys 315.

Research paper thumbnail of Inhibition of Class D β-Lactamases by Diaroyl Phosphates †

Biochemistry, 2005

The production of beta-lactamases is an important component of bacterial resistance to beta-lacta... more The production of beta-lactamases is an important component of bacterial resistance to beta-lactam antibiotics. These enzymes catalyze the hydrolytic destruction of beta-lactams. The class D serine beta-lactamases have, in recent years, been expanding in sequence space and substrate spectrum under the challenge of currently dispensed beta-lactams. Further, the beta-lactamase inhibitors now employed in medicine are not generally effective against class D enzymes. In this paper, we show that diaroyl phosphates are very effective inhibitory substrates of these enzymes. Reaction of the OXA-1 beta-lactamase, a typical class D enzyme, with diaroyl phosphates involves acylation of the active site with departure of an aroyl phosphate leaving group. The interaction of the latter with polar active-site residues is most likely responsible for the general reactivity of these molecules with the enzyme. The rate of acylation of the OXA-1 beta-lactamase by diaroyl phosphates is not greatly affected by the electronic effects of substituents, probably because of compensation phenomena, but is greatly enhanced by hydrophobic substituents; the second-order rate constant for acylation of the OXA-1 beta-lactamase by bis(4-phenylbenzoyl) phosphate, for example, is 1.1 x 10(7) s(-)(1) M(-)(1). This acylation reactivity correlates with the hydrophobic nature of the beta-lactam side-chain binding site of class D beta-lactamases. Deacylation of the enzyme is slow, e.g., 1.24 x 10(-)(3) s(-)(1) for the above-mentioned phosphate and directly influenced by the electronic effects of substituents. The effective steady-state inhibition constants, K(i), are nanomolar, e.g., 0.11 nM for the above-mentioned phosphate. The diaroyl phosphates, which have now been shown to be inhibitory substrates of all serine beta-lactamases, represent an intriguing new platform for the design of beta-lactamase inhibitors.

Research paper thumbnail of β-Secondary and Solvent Deuterium Kinetic Isotope Effects on Catalysis by the Streptomyces R61 DD-Peptidase:  Comparisons with a Structurally Similar Class C β-Lactamase †

Biochemistry, 1999

Secondary and solvent deuterium kinetic isotope effects have been determined for the steadystate ... more Secondary and solvent deuterium kinetic isotope effects have been determined for the steadystate kinetic parameters V/K and V for turnover of a depsipeptide substrate, m-[[(phenylacetyl)glycyl]oxy]benzoic acid, and of a-lactam substrate, penicillanic acid, by three typical class A-lactamases and a class C-lactamase. The isotope effects on alkaline hydrolysis of these substrates have been used as a frame of reference. The effect of the transition state conformation of the substrates in determining the-secondary isotope effects has been explicitly considered. The inverse-secondary isotope effects on both V/K and V for the class A enzymes with both substrates indicate transition states where the carbonyl group of the scissile bond has become tetrahedral and therefore reflect typical acyl-transfer transition states. The solvent isotope effects indicate that enzyme deacylation (as reflected in V for the Staphylococcus aureus PC1-lactamase) may be a classical general-base-catalyzed hydrolysis but that there is little proton motion in the enzyme acylation transition state (as revealed by V/K) for the TEM-lactamase and Bacillus cereus-lactamase I. These results provide kinetic support for the conjecture made on structural grounds that class A-lactamases employ an asymmetric double-displacement mechanism. The isotope effects on V/K for the class C-lactamase of Enterobacter cloacae P99 suggest an acyl-transfer transition state for the penicillin, although, as for the class A enzymes, without significant proton motion. On the other hand, the V/K transition state for depsipeptide does not seem to involve covalent chemistry. Suggestive of this conclusion are the measured-secondary isotope effect of 1.002 (0.012 and the inverse solvent isotope effect. These results provide an example of a significant difference between the kinetics of turnover of a-lactam and a depsipeptide by a-lactamase. The V transition state for both substrates with the P99-lactamase probably involves acyl-transfer (deacylation) where the conformation of the acyl-enzyme is closely restricted. The conformations of acyl-enzymes of the PC1 and P99-lactamases correlate to the (different) dispositions of general base catalysts at their active sites.

Research paper thumbnail of Inactivation of the Enterobacter cloacae P99 .beta.-Lactamase by a Fluorescent Phosphonate: Direct Detection of Ligand Binding at the Second Site

Biochemistry, 1995

The synthesis of a fluorescent beta-lactamase inhibitor, p-nitrophenyl [(dansylamido)methyl]-phos... more The synthesis of a fluorescent beta-lactamase inhibitor, p-nitrophenyl [(dansylamido)methyl]-phosphonate is described. The compound inactivated the class C beta-lactamase of Enterobacter cloacae P99 with stoichiometric release of p-nitrophenol, presumably, as with other phosphonate inhibitors, by phosphonylation of the active site serine. The inhibited enzyme exhibited typical dansyl fluorescence emission at 533 nm with excitation maxima at 345 and 283 nm; the latter excitation peak probably arises from radiationless energy transfer to the dansyl group from aromatic chromophores on the protein-inspection of the crystal structure shows that the closest are tyrosines. The fluorescence of the p-nitrophenyl phosphonate and the inhibited enzyme varied with pH in a very similar fashion, reflecting dissociation of the dimethylammonium ion in the ground state at low pH and of the sulfonamide in the excited state above pH 6. No perturbation of the fluorescence of the inhibited enzyme due to active site functional groups was observed. This may reflect the distance between the dansyl fluorophore and the phosphonyl group and/or the high pKa's of the protonated active site functional groups in the presence of the phosphonate. The addition of certain small molecular weight N-acyl amino acids, of preferred structure D-RCONHCHR'CO2-, to the inhibited enzyme led to an enhancement of dansyl fluorescence intensity and a blue shift in the emission maximum. This suggested that these molecules bind to the beta-lactamase at a site other than the active site and supports previous kinetic data to this effect [Dryjanski, M., & Pratt, R. F., (1995) Biochemistry 34, preceding paper in this issue].(ABSTRACT TRUNCATED AT 250 WORDS)

Research paper thumbnail of Crystallographic Structure of a Phosphonate Derivative of the Enterobacter cloacae P99 Cephalosporinase: Mechanistic Interpretation of a .beta.-Lactamase Transition-State Analog

Biochemistry, 1994

The crystal structure of a complex formed on reaction of the Enterobacter cloacae P99 cephalospor... more The crystal structure of a complex formed on reaction of the Enterobacter cloacae P99 cephalosporinase (p-lactamase) with a phosphonate monoester inhibitor, m-carboxyphenyl [ [N-[ (piodophenyl)acetyl]amino]methyl]phosphonate, has been obtained at 2.3-A resolution. The structure shows that the inhibitor has phosphonylated the active site serine (Ser64) with loss of the m-carboxyphenol leaving group. The inhibitor is positioned in the active site in a way that can be interpreted in terms of a transitionstate analog. The arylacetamido side chain is placed as anticipated from analogous P-lactamoyl complexes of penicillin-recognizing enzymes, with the amido group hydrogen-bonded to the backbone carbonyl of Ser3 18 (of the B3 P-strand) and to the amides of Gln 120 and Asnl52. There is support in the asymmetry of the hydrogen bonding of this side chain to the protein and in the 2-fold disorder of the benzyl group for the considerable breadth in substrate specificity exhibited by class C p-lactamases. One phosphonyl oxygen atom is in the oxyanion hole, hydrogen-bonded to main-chain NH groups of Ser3 18 and Ser64, while the other oxygen is solvated, not within hydrogen-bonding distance of any amino acid side chain. The closest active site functional group to the solvated oxygen atom is the Tyrl5O hydroxyl group (3.4 A); Lys67 and Lys3 15 are quite distant (4.3 and 5.7 A, respectively). Rather, Tyr 150 and Lys67 are more closely associated with Ser640y (2.9 and 3.3 A). This arrangement is interpreted in terms of the transition state for breakdown of the tetrahedral intermediate in the deacylation step of catalysis, where the Tyrl50 phenol seems the most likely general acid. Thus, Tyrl50, as the phenoxide anion, would be the general base catalyst in acylation, as proposed by Oefner et al. [ N a t u r e (1990) 343,284-2881. The structure is compared with that of a similar phosphonate derivative of a class A P-lactamase [Chen et al. (1993) J. Mol. B i d. 234, 165-1781, and mechanistic comparisons are made. The sensitivity of serine P-lactamases, as opposed to serine proteinases, toward inhibition by phosphonate monoanions is supported by electrostatic calculations showing a net positive potential only in the catalytic sites of the P-lactamases. The threat posed by bacterial infections to human health remains powerful because of the ever-increasing resistance of bacteria to the antibiotics that we deploy against them. A major source of resistance to the 0-lactam antibiotics is the ability of bacteria to produce enzymes, the p-lactamases, that specifically catalyze the hydrolysis of @-lactams (Neu, 1992; Liu et al., 1992). The most widespread and clinically important p-lactamases, of classes A and C (Waley, 1992), are serine hydrolases that catalyze p-lactam hydrolysis by a double displacement mechanism involving an acyl-enzyme intermediate. Our knowledge of these enzymes has been greatly advanced by the publication of crystal structures of both class A

Research paper thumbnail of Crystal Structures of Covalent Complexes of β-Lactam Antibiotics with Escherichia coli Penicillin-Binding Protein 5: Toward an Understanding of Antibiotic Specificity

Biochemistry, 2010

Penicillin-binding proteins (PBPs) are the molecular target for the widely used β-lactam class of... more Penicillin-binding proteins (PBPs) are the molecular target for the widely used β-lactam class of antibiotics, but how these compounds act at the molecular level is not fully understood. We have determined crystal structures of E. coli PBP5 as covalent complexes with imipenem, cloxacillin and cefoxitin. These antibiotics exhibit very different second order rates of acylation for the enzyme. In all three structures, there is excellent electron density for the central portion of the βlactam, but weak or absent density for the R1 or R2 side chains. Areas of contact between the antibiotics and PBP 5 do not correlate with the rates of acylation. The same is true for conformational changes because although shift of a loop leading to an electrostatic interaction between Arg248 and the β-lactam carboxylate, which occurs completely with cefoxitin, partially with imipenem and is absent with cloxacillin, is consistent with the different rates of acylation, mutagenesis of Arg248 only decreased cefoxitin acylation two fold. Together, these data suggest that structures of post-covalent complexes of PBP 5 are unlikely to be useful vehicles for design of new covalent inhibitors of PBPs. Finally, superimposition of the imipenem-acylated complex with PBP5 in complex with a boronic acid peptidemimetic shows that the position corresponding to the hydrolytic water molecule is occluded by the ring nitrogen of the β-lactam. Since the ring nitrogen occupies a similar position in all three complexes, this supports the hypothesis that deacylation is blocked by the continued presence of the leaving group after opening of the β-lactam ring. Penicillin-binding proteins (PBPs) are so named because they are the targets for the wellknown and widely used class of β-lactam antibiotics. Many PBPs are transpeptidases (TPases) that catalyze the formation of cross-links between peptides on adjacent strands of glycan during the final stages of peptidoglycan synthesis in bacteria, thereby conferring strength to the cell wall against osmotic pressure. Other PBPs are carboxypeptidases (CPases), which remove the terminal D-Ala of the pentapeptidyl substrate, or endopeptidases, † This work was supported by the National Institutes of Health grants GM66861 to CD, AI36901 to RAN and AI17986 to RFP.

Research paper thumbnail of Dipeptide Binding to the Extended Active Site of the Streptomyces R61 d -Alanyl- d -alanine-peptidase: The Path to a Specific Substrate †

Biochemistry, 2000

Bacterial cell walls are cross-linked in the final step of biosynthesis by specific D-alanyl-Dala... more Bacterial cell walls are cross-linked in the final step of biosynthesis by specific D-alanyl-Dalanine(DD)-peptidases/transpeptidases. The natural substrates of these enzymes should therefore be segments of peptidoglycan, but high specificity for such structures has yet to be demonstrated. The binding of dipeptides to the extended substrate binding site of the Streptomyces R61 DD-peptidase has been studied by means of a fluorescent-lactam probe. It was found that dipeptides of structure Gly-L-Xaa have affinity for a subsite adjacent to the-lactam binding site. Hydrophobic peptides such as Gly-L-Met and Gly-Laminocaprylic acid had the greatest affinity for this site, with dissociation constants in each case of 0.19 mM. A combination of this motif with the C-terminal D-alanyl-D-alanine moiety required of a DD-peptidase substrate yielded a new substrate, glycyl-L-R-amino-pimelyl-D-alanyl-D-alanine. Steady-state kinetic measurements established this compound as the most specific peptide substrate yet discovered for a DDpeptidase by at least 3 orders of magnitude (k cat) 69 s-1 , K m) 7.9 µM, k cat /K m) 8.7 × 10 6 s-1 M-1); acylation was rate-determining at saturation. This substrate, presumably not coincidentally, contains the acyl donor and acceptor moieties, appropriately separated, of the Streptomyces peptidoglycan structure. This general method of approach should be of value in the search for specific substrates and inhibitors (antibiotics) of other DD-peptidases.

Research paper thumbnail of Toward Better Antibiotics:  Crystallographic Studies of a Novel Class of DD-Peptidase/β-Lactamase Inhibitors †

Biochemistry, 2004

Lactam antibiotics are vital weapons in the treatment of bacterial infections, but their future i... more Lactam antibiotics are vital weapons in the treatment of bacterial infections, but their future is under increasing threat from-lactamases. These bacterial enzymes hydrolyze and inactivate-lactam antibiotics, rendering the host cell resistant to the bactericidal effects of the drugs. Nevertheless, the bacterial D-alanyl-D-alanine transpeptidases (DD-peptidases), the killing targets of-lactams, remain attractive targets for antibiotic compounds. Cyclic acyl phosph(on)ates have been developed and investigated as potential inhibitors of both transpeptidases and-lactamases. The X-ray crystal structures of the complexes of the Streptomyces strain R61 DD-peptidase inhibited by a bicyclic [1-hydroxy-4,5-benzo-2,6-dioxaphosphorinanone(3)-1-oxide] and a monocyclic [1-hydroxy-4-phenyl-2,6-dioxaphosphorinanone(3)-1-oxide] acyl phosphate were determined to investigate the mode of action of these novel inhibitors. The structures show, first, that these inhibitors form covalent bonds with the active site serine residue of the enzyme and that the refractory complexes thus formed are phosphoryl-enzyme species rather than acyl enzymes. The complexes are long-lived largely because, after ring opening, the ligands adopt conformations that cannot directly recyclize, the latter a phenomenon previously observed with cyclic acyl phosph(on)ates. While the two inhibitors bind in nearly identical conformations, the phosphoryl-enzyme complex formed from the monocyclic compound is significantly less mobile than that formed from the bicyclic compound. Despite this difference, the complex with the bicyclic compound breaks down to regenerate free enzyme somewhat more slowly than that of the monocyclic. This may be because of steric problems associated with the reorientation of the larger bicyclic ligand required for reactivation. The structures are strikingly different in the orientation of the phosphoryl moiety from those generated using more specific phosph(on)ates. Models of the noncovalent complexes of the monocyclic compound with the R61 DD-peptidase and a structurally very similar class C-lactamase suggest reasons why the former enzyme is phosphorylated by this compound, while the latter is acylated. Finally, this paper provides information that will help in the design of additional DD-peptidase inhibitors with the potential to serve as leads in the development of novel antibiotics.

Research paper thumbnail of The Crystal Structure of Phosphonate-Inhibited d-Ala-d-Ala Peptidase Reveals an Analogue of a Tetrahedral Transition State

Biochemistry, 2003

D-Alanyl-D-alanine carboxypeptidase/transpeptidases (DD-peptidases) are-lactam-sensitive enzymes ... more D-Alanyl-D-alanine carboxypeptidase/transpeptidases (DD-peptidases) are-lactam-sensitive enzymes that are responsible for the final peptidoglycan cross-linking step in bacterial cell wall biosynthesis. A highly specific tripeptide phosphonate inhibitor was designed with a side chain corresponding to a portion of the Streptomyces R61 peptidoglycan. This compound was found to be a slow, irreversible inactivator of the DD-peptidase. Molecular modeling suggested that although a pentacoordinated intermediate of the phosphonylation reaction would not interact strongly with the enzyme, a tetracoordinated phosphonyl enzyme might be analogous to a transition state in the reaction with peptide substrates. To investigate this possibility, the crystal structure of the phosphonyl enzyme was determined. The 1.1 Å resolution structure shows that the inhibitor has phosphonylated the catalytic serine (Ser62). One of the phosphonyl oxygens is noncovalently bound in the oxyanion hole, while the other is solvated by two water molecules. The conserved hydroxyl group of Tyr159 forms a strong hydrogen bond with the latter oxygen atom (2.77 Å). This arrangement is interpreted as being analogous to the transition state for the formation of the tetrahedral intermediate in the deacylation step of the carboxypeptidase reaction. The proximity of Tyr159 to the solvated phosphonyl oxygen suggests that the tyrosine anion acts as a general base for deacylation. This transition state analogue structure is compared to the structures of noncovalent DDpeptidase reaction intermediates and phosphonylated-lactamases. These comparisons show that specific substrate binding to the peptidase induces a conformational change in the active site that places Ser62 in an optimal position for catalysis. This activated conformation relaxes as the reaction proceeds.

Research paper thumbnail of β-Lactamase Inhibitors: Non-β-Lactams

Research paper thumbnail of The Perfect Penicillin? Inhibition of a Bacterial DD-Peptidase by Peptidoglycan-Mimetic β-Lactams

Journal of the American Chemical Society, 2004

Streptomyces R61 was a generous gift from Professor J.-M. Frère (University of Liège, Liège, Belg... more Streptomyces R61 was a generous gift from Professor J.-M. Frère (University of Liège, Liège, Belgium). Lα-aminopimelic acid was synthesized as described by Rosowsky et al. S1 The substrate 1 was prepared by Dr. John W. Anderson in this laboratory S2 ; the synthesis of 4 will be described in a separate publication. All other reagents were from commercial sources and used as received. 1 H NMR † spectra were obtained from a Varian Gemini-300 MHz spectrometer. IR spectra were recorded on a Perkin Elmer 1600 FTIR instrument. Preparative thin layer chromatography (TLC) was performed on Analtech 20x20 cm, 2000micron F254 plates. The plates were visualized with UV light. Electrospray mass spectra were obtained from the

Research paper thumbnail of Synthesis and Evaluation of New Substrate Analogues of Streptomyces R61 dd -Peptidase:  Dissection of a Specific Ligand

The Journal of Organic Chemistry, 2004

Good substrates of the Streptomyces R61 dd-peptidase, such as glycyl-L-alpha-amino-epsilon-pimely... more Good substrates of the Streptomyces R61 dd-peptidase, such as glycyl-L-alpha-amino-epsilon-pimelyl-D-alanyl-D-alanine, 1 (Anderson, J. W.; Pratt, R. F. Biochemistry 2000, 39, 12200-12209), contain the glycyl-L-alpha-amino-epsilon-pimelyl side chain. A number of thia variants of this structure have been synthesized by means of a disconnection strategy whereby the appropriate thiols were reacted with either acryloyl-D-alanyl-D-alanine or haloalkanoyl-D-alanyl-D-alanines. Kinetics studies of the hydrolysis of these compounds by the R61 DD-peptidase showed that the presence of the N-terminal glycylammonium ion and the pimelyl-alpha-carboxylate are very important for efficient catalysis. The results of deletion of the C-terminal D-alanine indicate a promising direction toward new inhibitors. Shorter (one methylene less) and longer (one methylene more) analogues of 1 are also poor substrates. Molecular modeling and dynamics studies suggest that the higher mobility of the active site residues and the modified substrates in enzyme-substrate complexes may be the dominant factor in this loss of reactivity. The general conclusion is that essentially all of the structural elements of the side chain of 1 are required to produce a good substrate. This result has important implications for the design of inhibitors of DD-peptidases.

Research paper thumbnail of Crystal Structure of the Bacillus subtilis Penicillin-binding Protein 4a, and its Complex with a Peptidoglycan Mimetic Peptide

Journal of Molecular Biology, 2007

The genome of Bacillus subtilis encodes 16 penicillin-binding proteins (PBPs) involved in the syn... more The genome of Bacillus subtilis encodes 16 penicillin-binding proteins (PBPs) involved in the synthesis and/or remodelling of the peptidoglycan during the complex life cycle of this sporulating Gram-positive rod-shaped bacterium. PBP4a (encoded by the dacC gene) is a low-molecular mass PBP clearly exhibiting in vitro DD-carboxypeptidase activity. We have solved the crystal structure of this protein alone and in complex with a peptide (D-αaminopymelyl-ε-D-alanyl-D-alanine) that mimics the C-terminal end of the Bacillus peptidoglycan stem peptide. PBP4a is composed of three domains: the penicillin-binding domain with a fold similar to the class A β-lactamase structure and two domains inserted between the conserved motifs 1 and 2 characteristic of the penicillin-recognizing enzymes. The soaking of PBP4a in a solution of D-α-aminopymelyl-ε-D-alanyl-D-alanine resulted in an adduct between PBP4a and a D-α-aminopimelyl-ε-D-alanine dipeptide and an unbound D-alanine, i.e. the products of acylation of PBP4a by D-α-aminopymelyl-ε-D-alanyl-D-alanine with the release of a D-alanine. The adduct also reveals a binding pocket specific to the diaminopimelic acid, the third residue of the peptidoglycan stem pentapeptide of B. subtilis. This pocket is specific for this class of PBPs.

Research paper thumbnail of Structures of Two Kinetic Intermediates Reveal Species Specificity of Penicillin-binding Proteins

Journal of Molecular Biology, 2002

Research paper thumbnail of Crystal Structures of Complexes of Bacterial dd-Peptidases with Peptidoglycan-Mimetic Ligands: The Substrate Specificity Puzzle

Journal of Molecular Biology, 2008

The X-ray crystal structures of covalent complexes of the Actinomadura R39 DD-peptidase and Esche... more The X-ray crystal structures of covalent complexes of the Actinomadura R39 DD-peptidase and Escherichia coli penicillin-binding protein (PBP) 5 with βlactams bearing peptidoglycan-mimetic side chains have been determined. The structure of the hydrolysis product of an analogous peptide bound noncovalently to the former enzyme has also been obtained. The R39 DDpeptidase structures reveal the presence of a specific binding site for the D-αaminopimelyl side chain, characteristic of the stem peptide of Actinomadura R39. This binding site features a hydrophobic cleft for the pimelyl methylene groups and strong hydrogen bonding to the polar terminus. Both of these active site elements are provided by amino acid side chains from two separate domains of the protein. In contrast, no clear electron density corresponding to the terminus of the peptidoglycan-mimetic side chains is present when these β-lactams are covalently bound to PBP5. There is, therefore, no indication of a specific side-chain binding site in this enzyme. These results are in agreement with those from kinetics studies published earlier and support the general prediction made at the time of a direct correlation between kinetics and structural evidence. The essential highmolecular-mass PBPs have demonstrated, to date, no specific reactivity with peptidoglycan-mimetic peptide substrates and β-lactam inhibitors and, thus, probably do not possess a specific substrate-binding site of the type demonstrated here with the R39 DD-peptidase. This striking deficiency may represent a sophisticated defense mechanism against low-molecular-mass substrate-analogue inhibitors/antibiotics; its discovery should focus new inhibitor design.

Research paper thumbnail of Formation and Structure of 1:1 Complexes between Aryl Hydroxamic Acids and Vanadate at Neutral pH

Inorganic Chemistry

Although aryl hydroxamic acids are well-known to form coordination complexes with vanadate (V V),... more Although aryl hydroxamic acids are well-known to form coordination complexes with vanadate (V V), the nature of these complexes at neutral pH and submillimolar concentrations, the conditions under which such complexes inhibit various serine amidohydrolases, is not well established. A series of qualitative and quantitative experiments, involving UV/vis, 1 H NMR, and 51 V NMR spectroscopies, established that both 1:1 and 1:2 vanadate/hydroxamate complexes form at pH 7.5, with the former dominating at submillimolar concentrations. Formation constants for the complexes of several aryl and alkyl hydroxamic acids were determined; for example, for benzohydroxamic acid, the stepwise formation constants of the 1:1 and 1:2 complexes were 3000 and 400 M-1 , respectively. The 51 V chemical shift of the 1:1 4-nitrobenzohydroxamic acid complex was −497 ppm, and that of its unsubstituted analogue was −498 ppm. A 1 H− 15 N HSQC spectrum of the 4-nitrobenzo-15 N-hydroxamic acid/vanadate complex indicated the presence of an N−H group with 15 N and 1 H chemical shifts of 115 and 5.83 ppm, respectively. A 13 C NMR spectrum of the complex of 4-nitrobenzo-13 C-hydroxamic acid with vanadate displayed a resonance at 170.1 ppm and thus a coordination-induced shift (CIS) of +3.8 ppm. In contrast, the CIS value of an established 1:2 complex, thought to contain chelated hydroxamic acid ligands, was +11.9 ppm. These spectral data led to the following structural picture of 1:1 complexes of vanadate and aryl hydroxamic acids. They contain penta-or hexa-coordinated vanadium. The ligand is in the hydroxamate rather than hydroximate form. The ligand is presumably bound to vanadium through the hydroxamic hydroxyl oxygen, but the hydroxamic acid carbonyl oxygen interacts weakly with vanadium. These species are the most likely candidates for the inhibitors of serine amidohydrolases found in vanadate/ hydroxamic acid mixtures.

Research paper thumbnail of Benzopyranones with retro-amide side chains as (inhibitory) β-lactamase substrates

Bioorganic & Medicinal Chemistry Letters, 2004

Benzopyran derivatives R 0350 Benzopyranones with Retro-Amide Side Chains as (Inhibitory) β-Lacta... more Benzopyran derivatives R 0350 Benzopyranones with Retro-Amide Side Chains as (Inhibitory) β-Lactamase Substrates.-3-Carbamoylbenzopyranones (I) are synthesized (no yields are given) and found to be comparably effective as substrates of typical class A, C and D β-lactamases as analogous benzopyranones bearing 'normal' amide side chains. The new 8-carboxy derivative (Ib), however, forms a much more tightly-bound acyl-enzyme with a class C β-lactamase compared to its 'normal' analogue, and thus provides a structural lead to new inhibitors of this class of β-lactamase.-(ADEDIRAN, S. A.; CABARET, D.;

Research paper thumbnail of Synthesis and β-lactamase reactivity of α-substituted phenaceturates

Bioorganic & Medicinal Chemistry, 2006

Lactams with 6a (penicillins) or 7a (cephalosporins) substituents are often b-lactamase inhibitor... more Lactams with 6a (penicillins) or 7a (cephalosporins) substituents are often b-lactamase inhibitors. This paper assesses the effect of such substituents on acyclic b-lactamase substrates. Thus, a series of m-carboxyphenyl phenaceturates, substituted at the glycyl a-carbon by-OMe,-CH 2 OH,-CO 2 À , and-CH 2 NH 3 + , have been prepared, and tested for their reactivity against serine b-lactamases. The latter two are novel substituents in b-lactamase substrates. The methoxy and hydroxymethyl compounds were found to be poor to moderately good substrates, depending on the enzyme. The aminomethyl compound gave rise to a transiently stable (t 1/2 = 4.6 s) complex on its reaction with a class C b-lactamase. The reactivity of the compounds against three low molecular weight DD DD-peptidases was also tested. Again, the methoxy and hydroxymethyl compounds proved to be quite good substrates with no sign of inhibitory complexes. The DD DD-peptidases reacted with one enantiomer (the compounds were prepared as racemates), presumably the D D compound. The class C b-lactamase reacted with both D D and L L enantiomers although it preferred the latter. The structural bases of these stereo-preferences were explored by reference to the crystal structure of the enzyme by molecular modeling studies. The aminomethyl compound was unreactive with the DD DDpeptidases, whereas the carboxy compound did not react with any of the above-mentioned enzymes. The inhibitory effects of the-OMe and-CH 2 OH substituents in b-lactams apparently require a combination of the substituent and the pendant leaving group of the b-lactam at the acyl-enzyme stage.

Research paper thumbnail of Inhibition of Class A and C β-Lactamases by Diaroyl Phosphates

Biochemistry, 2009

A series of diaroyl phosphates was employed to assess the general reactivity of this class of mol... more A series of diaroyl phosphates was employed to assess the general reactivity of this class of molecule against classical class A and class C beta-lactamases. The compounds were found, in general, to be inhibitory substrates of both classes of enzyme. In each case, they reacted rapidly with the enzyme (10(4) to 10(6) s(-1) M(-1)) to yield transiently stable intermediates, most likely acyl-enzymes, which slowly (10(-3) to 10(-1) s(-1)) regenerated free enzyme. In certain cases, side branches from direct turnover produced EII complexes ("substrate" inhibition), more inert EI' complexes, and, in one case, a completely inactive EI' complex. Deacylation, but not acylation, was enhanced by electron-withdrawing substituents. Acylation rates were enhanced by hydrophobic substitution, both in the diaroyl phosphate and at the enzyme active site. The latter factor led to the general order of beta-lactamase acylation rates: class D (previous results) > class C > class A. It is likely that nanomolar inhibitors of all serine beta-lactamases could be achieved by rational exploitation of diacyl phosphates.

Research paper thumbnail of Intramolecular Cooperativity in the Reaction of Diacyl Phosphates with Serine β-Lactamases

Biochemistry, 2009

Asymmetric diaroyl phosphates (ArCOOPO 2 − OCOAr′, where Ar = Ph, Ar′ = 4-biphenyl, 2benzothiophe... more Asymmetric diaroyl phosphates (ArCOOPO 2 − OCOAr′, where Ar = Ph, Ar′ = 4-biphenyl, 2benzothiophenyl and 2-benzofuranyl), have been prepared, evaluated as serine (classes A, C, D) βlactamase inhibitors, and compared with respect to the latter with their symmetric "parents", where Ar = Ar′. The asymmetric compounds, in general, were found to react with the β-lactamases in two modes, corresponding to different orientations with respect to the active site, whereby either of the two aroyl groups may acylate the enzyme to form two different inert acyl-enzymes, E-COAr and E-COAr′. In all cases, the asymmetric compounds, in one orientation, react more rapidly with the enzymes tested than one symmetrical "parent" but not both. From comparisons of activation free energy differences, it was found that the changes in free energy on changing from one aryl group to another, in either the acyl group or the leaving group, were not additive, i.e. that the effect of changing one aroyl group to another depended on the leaving group and vice versa. Thus, intramolecular cooperativity between the aroyl groups must exist, arising either from direct interaction between them or from protein-mediated interaction, or from a combination of both. Such cooperativity brings fresh opportunities and challenges to the search for novel ß-lactamase inhibitors. Aroyl phosphates, 1, have proven to be very effective inhibitors of serine ß-lactamases (1-4). The phosphate leaving group appears to interact with polar resides in a way as to enhance active site acylation by both specific amidoacyl groups and non-classical aroyl groups (2-4,5,6). Acylation by the latter leads to hydrolytically refractive acyl-enzymes and thus to significant inhibition (Scheme 1). A series of diaroyl phosphates 2 have proven to be particularly effective inhibitors (3,4). Structure-activity studies have shown that hydrophobic substituents entrance inhibition, largely through enhancement of acylation rates, while electron-donating substituents enhance inhibition by depression of deacylation rates (3).

Research paper thumbnail of Mechanism of Reaction of Acyl Phosph(on)ates with the β-Lactamase of Enterobacter cloacae P99

Biochemistry

A series of acyl phosph(on)ates has been prepared to more closely examine the details of the inte... more A series of acyl phosph(on)ates has been prepared to more closely examine the details of the interactions of this class of molecule with beta-lactamases. In general, they were found to react with the class C beta-lactamase of Enterobacter cloacae P99 in two ways, by acylation and by phosphylation. The acyl-enzymes generated by the former reaction were transiently stable with half-lives of between 3 and 45 s, of comparable lifetime therefore to those generated by the inhibitory beta-lactams cefotaxime, cefuroxime, and cefoxitin. On the other hand, phosphylation led to a completely inactive enzyme. In general, the second-order rate constants for acylation (k(cat)/K(m)) were larger than for phosphylation (k(i)). As expected on chemical grounds, phosphylation was found to be relatively more effective for the phosphonates than the phosphates. The acyl phosphates were much more effective acylating agents however. The acylation reaction was found to be enhanced by hydrophobic substituents in both the acyl and leaving group moieties. Thus, the most reactive compound in this series was benzo[b]thiophene-2-carbonyl 2'-naphthyl phosphate with a K(m) value of 0.15 microM and a k(cat) of 0.2 s(-1); k(cat)/K(m) is therefore 1.3 x 10(6) s(-1) M(-1), making this compound the most specific acyclic acylation reagent for this beta-lactamase yet described. Significant substrate inhibition by this compound suggested that further binding regions may be available for exploitation in inhibitor design. A linear free energy analysis showed that the transition states for acylation of the beta-lactamase by aroyl phosphates are analogues of the corresponding aryl boronic acid adducts. Molecular modeling suggested that the aroyl phosphates react with the P99 beta-lactamase with the aroyl group in the side chain/acyl group site of normal substrates and the phosphate in the leaving group site. In this orientation, the phosphate leaving group interacts strongly with Lys 315.

Research paper thumbnail of Inhibition of Class D β-Lactamases by Diaroyl Phosphates †

Biochemistry, 2005

The production of beta-lactamases is an important component of bacterial resistance to beta-lacta... more The production of beta-lactamases is an important component of bacterial resistance to beta-lactam antibiotics. These enzymes catalyze the hydrolytic destruction of beta-lactams. The class D serine beta-lactamases have, in recent years, been expanding in sequence space and substrate spectrum under the challenge of currently dispensed beta-lactams. Further, the beta-lactamase inhibitors now employed in medicine are not generally effective against class D enzymes. In this paper, we show that diaroyl phosphates are very effective inhibitory substrates of these enzymes. Reaction of the OXA-1 beta-lactamase, a typical class D enzyme, with diaroyl phosphates involves acylation of the active site with departure of an aroyl phosphate leaving group. The interaction of the latter with polar active-site residues is most likely responsible for the general reactivity of these molecules with the enzyme. The rate of acylation of the OXA-1 beta-lactamase by diaroyl phosphates is not greatly affected by the electronic effects of substituents, probably because of compensation phenomena, but is greatly enhanced by hydrophobic substituents; the second-order rate constant for acylation of the OXA-1 beta-lactamase by bis(4-phenylbenzoyl) phosphate, for example, is 1.1 x 10(7) s(-)(1) M(-)(1). This acylation reactivity correlates with the hydrophobic nature of the beta-lactam side-chain binding site of class D beta-lactamases. Deacylation of the enzyme is slow, e.g., 1.24 x 10(-)(3) s(-)(1) for the above-mentioned phosphate and directly influenced by the electronic effects of substituents. The effective steady-state inhibition constants, K(i), are nanomolar, e.g., 0.11 nM for the above-mentioned phosphate. The diaroyl phosphates, which have now been shown to be inhibitory substrates of all serine beta-lactamases, represent an intriguing new platform for the design of beta-lactamase inhibitors.

Research paper thumbnail of β-Secondary and Solvent Deuterium Kinetic Isotope Effects on Catalysis by the Streptomyces R61 DD-Peptidase:  Comparisons with a Structurally Similar Class C β-Lactamase †

Biochemistry, 1999

Secondary and solvent deuterium kinetic isotope effects have been determined for the steadystate ... more Secondary and solvent deuterium kinetic isotope effects have been determined for the steadystate kinetic parameters V/K and V for turnover of a depsipeptide substrate, m-[[(phenylacetyl)glycyl]oxy]benzoic acid, and of a-lactam substrate, penicillanic acid, by three typical class A-lactamases and a class C-lactamase. The isotope effects on alkaline hydrolysis of these substrates have been used as a frame of reference. The effect of the transition state conformation of the substrates in determining the-secondary isotope effects has been explicitly considered. The inverse-secondary isotope effects on both V/K and V for the class A enzymes with both substrates indicate transition states where the carbonyl group of the scissile bond has become tetrahedral and therefore reflect typical acyl-transfer transition states. The solvent isotope effects indicate that enzyme deacylation (as reflected in V for the Staphylococcus aureus PC1-lactamase) may be a classical general-base-catalyzed hydrolysis but that there is little proton motion in the enzyme acylation transition state (as revealed by V/K) for the TEM-lactamase and Bacillus cereus-lactamase I. These results provide kinetic support for the conjecture made on structural grounds that class A-lactamases employ an asymmetric double-displacement mechanism. The isotope effects on V/K for the class C-lactamase of Enterobacter cloacae P99 suggest an acyl-transfer transition state for the penicillin, although, as for the class A enzymes, without significant proton motion. On the other hand, the V/K transition state for depsipeptide does not seem to involve covalent chemistry. Suggestive of this conclusion are the measured-secondary isotope effect of 1.002 (0.012 and the inverse solvent isotope effect. These results provide an example of a significant difference between the kinetics of turnover of a-lactam and a depsipeptide by a-lactamase. The V transition state for both substrates with the P99-lactamase probably involves acyl-transfer (deacylation) where the conformation of the acyl-enzyme is closely restricted. The conformations of acyl-enzymes of the PC1 and P99-lactamases correlate to the (different) dispositions of general base catalysts at their active sites.

Research paper thumbnail of Inactivation of the Enterobacter cloacae P99 .beta.-Lactamase by a Fluorescent Phosphonate: Direct Detection of Ligand Binding at the Second Site

Biochemistry, 1995

The synthesis of a fluorescent beta-lactamase inhibitor, p-nitrophenyl [(dansylamido)methyl]-phos... more The synthesis of a fluorescent beta-lactamase inhibitor, p-nitrophenyl [(dansylamido)methyl]-phosphonate is described. The compound inactivated the class C beta-lactamase of Enterobacter cloacae P99 with stoichiometric release of p-nitrophenol, presumably, as with other phosphonate inhibitors, by phosphonylation of the active site serine. The inhibited enzyme exhibited typical dansyl fluorescence emission at 533 nm with excitation maxima at 345 and 283 nm; the latter excitation peak probably arises from radiationless energy transfer to the dansyl group from aromatic chromophores on the protein-inspection of the crystal structure shows that the closest are tyrosines. The fluorescence of the p-nitrophenyl phosphonate and the inhibited enzyme varied with pH in a very similar fashion, reflecting dissociation of the dimethylammonium ion in the ground state at low pH and of the sulfonamide in the excited state above pH 6. No perturbation of the fluorescence of the inhibited enzyme due to active site functional groups was observed. This may reflect the distance between the dansyl fluorophore and the phosphonyl group and/or the high pKa's of the protonated active site functional groups in the presence of the phosphonate. The addition of certain small molecular weight N-acyl amino acids, of preferred structure D-RCONHCHR'CO2-, to the inhibited enzyme led to an enhancement of dansyl fluorescence intensity and a blue shift in the emission maximum. This suggested that these molecules bind to the beta-lactamase at a site other than the active site and supports previous kinetic data to this effect [Dryjanski, M., & Pratt, R. F., (1995) Biochemistry 34, preceding paper in this issue].(ABSTRACT TRUNCATED AT 250 WORDS)

Research paper thumbnail of Crystallographic Structure of a Phosphonate Derivative of the Enterobacter cloacae P99 Cephalosporinase: Mechanistic Interpretation of a .beta.-Lactamase Transition-State Analog

Biochemistry, 1994

The crystal structure of a complex formed on reaction of the Enterobacter cloacae P99 cephalospor... more The crystal structure of a complex formed on reaction of the Enterobacter cloacae P99 cephalosporinase (p-lactamase) with a phosphonate monoester inhibitor, m-carboxyphenyl [ [N-[ (piodophenyl)acetyl]amino]methyl]phosphonate, has been obtained at 2.3-A resolution. The structure shows that the inhibitor has phosphonylated the active site serine (Ser64) with loss of the m-carboxyphenol leaving group. The inhibitor is positioned in the active site in a way that can be interpreted in terms of a transitionstate analog. The arylacetamido side chain is placed as anticipated from analogous P-lactamoyl complexes of penicillin-recognizing enzymes, with the amido group hydrogen-bonded to the backbone carbonyl of Ser3 18 (of the B3 P-strand) and to the amides of Gln 120 and Asnl52. There is support in the asymmetry of the hydrogen bonding of this side chain to the protein and in the 2-fold disorder of the benzyl group for the considerable breadth in substrate specificity exhibited by class C p-lactamases. One phosphonyl oxygen atom is in the oxyanion hole, hydrogen-bonded to main-chain NH groups of Ser3 18 and Ser64, while the other oxygen is solvated, not within hydrogen-bonding distance of any amino acid side chain. The closest active site functional group to the solvated oxygen atom is the Tyrl5O hydroxyl group (3.4 A); Lys67 and Lys3 15 are quite distant (4.3 and 5.7 A, respectively). Rather, Tyr 150 and Lys67 are more closely associated with Ser640y (2.9 and 3.3 A). This arrangement is interpreted in terms of the transition state for breakdown of the tetrahedral intermediate in the deacylation step of catalysis, where the Tyrl50 phenol seems the most likely general acid. Thus, Tyrl50, as the phenoxide anion, would be the general base catalyst in acylation, as proposed by Oefner et al. [ N a t u r e (1990) 343,284-2881. The structure is compared with that of a similar phosphonate derivative of a class A P-lactamase [Chen et al. (1993) J. Mol. B i d. 234, 165-1781, and mechanistic comparisons are made. The sensitivity of serine P-lactamases, as opposed to serine proteinases, toward inhibition by phosphonate monoanions is supported by electrostatic calculations showing a net positive potential only in the catalytic sites of the P-lactamases. The threat posed by bacterial infections to human health remains powerful because of the ever-increasing resistance of bacteria to the antibiotics that we deploy against them. A major source of resistance to the 0-lactam antibiotics is the ability of bacteria to produce enzymes, the p-lactamases, that specifically catalyze the hydrolysis of @-lactams (Neu, 1992; Liu et al., 1992). The most widespread and clinically important p-lactamases, of classes A and C (Waley, 1992), are serine hydrolases that catalyze p-lactam hydrolysis by a double displacement mechanism involving an acyl-enzyme intermediate. Our knowledge of these enzymes has been greatly advanced by the publication of crystal structures of both class A

Research paper thumbnail of Crystal Structures of Covalent Complexes of β-Lactam Antibiotics with Escherichia coli Penicillin-Binding Protein 5: Toward an Understanding of Antibiotic Specificity

Biochemistry, 2010

Penicillin-binding proteins (PBPs) are the molecular target for the widely used β-lactam class of... more Penicillin-binding proteins (PBPs) are the molecular target for the widely used β-lactam class of antibiotics, but how these compounds act at the molecular level is not fully understood. We have determined crystal structures of E. coli PBP5 as covalent complexes with imipenem, cloxacillin and cefoxitin. These antibiotics exhibit very different second order rates of acylation for the enzyme. In all three structures, there is excellent electron density for the central portion of the βlactam, but weak or absent density for the R1 or R2 side chains. Areas of contact between the antibiotics and PBP 5 do not correlate with the rates of acylation. The same is true for conformational changes because although shift of a loop leading to an electrostatic interaction between Arg248 and the β-lactam carboxylate, which occurs completely with cefoxitin, partially with imipenem and is absent with cloxacillin, is consistent with the different rates of acylation, mutagenesis of Arg248 only decreased cefoxitin acylation two fold. Together, these data suggest that structures of post-covalent complexes of PBP 5 are unlikely to be useful vehicles for design of new covalent inhibitors of PBPs. Finally, superimposition of the imipenem-acylated complex with PBP5 in complex with a boronic acid peptidemimetic shows that the position corresponding to the hydrolytic water molecule is occluded by the ring nitrogen of the β-lactam. Since the ring nitrogen occupies a similar position in all three complexes, this supports the hypothesis that deacylation is blocked by the continued presence of the leaving group after opening of the β-lactam ring. Penicillin-binding proteins (PBPs) are so named because they are the targets for the wellknown and widely used class of β-lactam antibiotics. Many PBPs are transpeptidases (TPases) that catalyze the formation of cross-links between peptides on adjacent strands of glycan during the final stages of peptidoglycan synthesis in bacteria, thereby conferring strength to the cell wall against osmotic pressure. Other PBPs are carboxypeptidases (CPases), which remove the terminal D-Ala of the pentapeptidyl substrate, or endopeptidases, † This work was supported by the National Institutes of Health grants GM66861 to CD, AI36901 to RAN and AI17986 to RFP.

Research paper thumbnail of Dipeptide Binding to the Extended Active Site of the Streptomyces R61 d -Alanyl- d -alanine-peptidase: The Path to a Specific Substrate †

Biochemistry, 2000

Bacterial cell walls are cross-linked in the final step of biosynthesis by specific D-alanyl-Dala... more Bacterial cell walls are cross-linked in the final step of biosynthesis by specific D-alanyl-Dalanine(DD)-peptidases/transpeptidases. The natural substrates of these enzymes should therefore be segments of peptidoglycan, but high specificity for such structures has yet to be demonstrated. The binding of dipeptides to the extended substrate binding site of the Streptomyces R61 DD-peptidase has been studied by means of a fluorescent-lactam probe. It was found that dipeptides of structure Gly-L-Xaa have affinity for a subsite adjacent to the-lactam binding site. Hydrophobic peptides such as Gly-L-Met and Gly-Laminocaprylic acid had the greatest affinity for this site, with dissociation constants in each case of 0.19 mM. A combination of this motif with the C-terminal D-alanyl-D-alanine moiety required of a DD-peptidase substrate yielded a new substrate, glycyl-L-R-amino-pimelyl-D-alanyl-D-alanine. Steady-state kinetic measurements established this compound as the most specific peptide substrate yet discovered for a DDpeptidase by at least 3 orders of magnitude (k cat) 69 s-1 , K m) 7.9 µM, k cat /K m) 8.7 × 10 6 s-1 M-1); acylation was rate-determining at saturation. This substrate, presumably not coincidentally, contains the acyl donor and acceptor moieties, appropriately separated, of the Streptomyces peptidoglycan structure. This general method of approach should be of value in the search for specific substrates and inhibitors (antibiotics) of other DD-peptidases.

Research paper thumbnail of Toward Better Antibiotics:  Crystallographic Studies of a Novel Class of DD-Peptidase/β-Lactamase Inhibitors †

Biochemistry, 2004

Lactam antibiotics are vital weapons in the treatment of bacterial infections, but their future i... more Lactam antibiotics are vital weapons in the treatment of bacterial infections, but their future is under increasing threat from-lactamases. These bacterial enzymes hydrolyze and inactivate-lactam antibiotics, rendering the host cell resistant to the bactericidal effects of the drugs. Nevertheless, the bacterial D-alanyl-D-alanine transpeptidases (DD-peptidases), the killing targets of-lactams, remain attractive targets for antibiotic compounds. Cyclic acyl phosph(on)ates have been developed and investigated as potential inhibitors of both transpeptidases and-lactamases. The X-ray crystal structures of the complexes of the Streptomyces strain R61 DD-peptidase inhibited by a bicyclic [1-hydroxy-4,5-benzo-2,6-dioxaphosphorinanone(3)-1-oxide] and a monocyclic [1-hydroxy-4-phenyl-2,6-dioxaphosphorinanone(3)-1-oxide] acyl phosphate were determined to investigate the mode of action of these novel inhibitors. The structures show, first, that these inhibitors form covalent bonds with the active site serine residue of the enzyme and that the refractory complexes thus formed are phosphoryl-enzyme species rather than acyl enzymes. The complexes are long-lived largely because, after ring opening, the ligands adopt conformations that cannot directly recyclize, the latter a phenomenon previously observed with cyclic acyl phosph(on)ates. While the two inhibitors bind in nearly identical conformations, the phosphoryl-enzyme complex formed from the monocyclic compound is significantly less mobile than that formed from the bicyclic compound. Despite this difference, the complex with the bicyclic compound breaks down to regenerate free enzyme somewhat more slowly than that of the monocyclic. This may be because of steric problems associated with the reorientation of the larger bicyclic ligand required for reactivation. The structures are strikingly different in the orientation of the phosphoryl moiety from those generated using more specific phosph(on)ates. Models of the noncovalent complexes of the monocyclic compound with the R61 DD-peptidase and a structurally very similar class C-lactamase suggest reasons why the former enzyme is phosphorylated by this compound, while the latter is acylated. Finally, this paper provides information that will help in the design of additional DD-peptidase inhibitors with the potential to serve as leads in the development of novel antibiotics.

Research paper thumbnail of The Crystal Structure of Phosphonate-Inhibited d-Ala-d-Ala Peptidase Reveals an Analogue of a Tetrahedral Transition State

Biochemistry, 2003

D-Alanyl-D-alanine carboxypeptidase/transpeptidases (DD-peptidases) are-lactam-sensitive enzymes ... more D-Alanyl-D-alanine carboxypeptidase/transpeptidases (DD-peptidases) are-lactam-sensitive enzymes that are responsible for the final peptidoglycan cross-linking step in bacterial cell wall biosynthesis. A highly specific tripeptide phosphonate inhibitor was designed with a side chain corresponding to a portion of the Streptomyces R61 peptidoglycan. This compound was found to be a slow, irreversible inactivator of the DD-peptidase. Molecular modeling suggested that although a pentacoordinated intermediate of the phosphonylation reaction would not interact strongly with the enzyme, a tetracoordinated phosphonyl enzyme might be analogous to a transition state in the reaction with peptide substrates. To investigate this possibility, the crystal structure of the phosphonyl enzyme was determined. The 1.1 Å resolution structure shows that the inhibitor has phosphonylated the catalytic serine (Ser62). One of the phosphonyl oxygens is noncovalently bound in the oxyanion hole, while the other is solvated by two water molecules. The conserved hydroxyl group of Tyr159 forms a strong hydrogen bond with the latter oxygen atom (2.77 Å). This arrangement is interpreted as being analogous to the transition state for the formation of the tetrahedral intermediate in the deacylation step of the carboxypeptidase reaction. The proximity of Tyr159 to the solvated phosphonyl oxygen suggests that the tyrosine anion acts as a general base for deacylation. This transition state analogue structure is compared to the structures of noncovalent DDpeptidase reaction intermediates and phosphonylated-lactamases. These comparisons show that specific substrate binding to the peptidase induces a conformational change in the active site that places Ser62 in an optimal position for catalysis. This activated conformation relaxes as the reaction proceeds.