Crystal structures of penicillin acylase enzyme-substrate complexes: structural insights into the catalytic mechanism (original) (raw)
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Journal of Molecular Biology, 2001
The crystal structure of penicillin G acylase from Escherichia coli has been determined to a resolution of 1.3 A Ê from a crystal form grown in the presence of ethylene glycol. To study aspects of the substrate speci®city and catalytic mechanism of this key biotechnological enzyme, mutants were made to generate inactive protein useful for producing enzyme-substrate complexes. Owing to the intimate association of enzyme activity and precursor processing in this protein family (the Ntn hydrolases), most attempts to alter active-site residues lead to processing defects. Mutation of the invariant residue Arg B263 results in the accumulation of a protein precursor form. However, the mutation of Asn B241, a residue implicated in stabilisation of the tetrahedral intermediate during catalysis, inactivates the enzyme but does not prevent autocatalytic processing or the ability to bind substrates. The crystal structure of the Asn B241 Ala oxyanion hole mutant enzyme has been determined in its native form and in complex with penicillin G and penicillin G sulphoxide. We show that Asn B241 has an important role in maintaining the active site geometry and in productive substrate binding, hence the structure of the mutant protein is a poor model for the Michaelis complex. For this reason, we subsequently solved the structure of the wild-type protein in complex with the slowly processed substrate penicillin G sulphoxide. Analysis of this structure suggests that the reaction mechanism proceeds via direct nucleophilic attack of Ser B1 on the scissile amide and not as previously proposed via a tightly H-bonded water molecule acting as a``virtual'' base.
Structural Studies of Penicillin Acylase
Applied Biochemistry and Biotechnology, 2000
Penicillin acylases are used in the pharmaceutical industry for the preparation of antibiotics. The 3-D structure of Penicillin G acylase from Escherichia coli has been solved. Here, we present structural data that pertain to the unanswered questions that arose from the original strucutre. Specificity for the amide portion of substrate was probed by the structure determination of a range of complexes with substitutions around the phenylacetyl ring of the ligand. Altered substrate specificity mutations derived from an in vivo positive selection process have also been studied, revealing the structural consequences of mutation at position B71. Protein processing has been analyzed by the construction of site-directed mutants, which affect this reaction with two distinct phenotypes. Mutations that allow processing but yield inactive protein provide the structure of an ES complex with a true substrate, with implications for the enzymatic mechanism and stereospecificity of the reaction. Mutations that preclude processing have allowed the structure of the precursor, which includes the 54 amino acid linker region normally removed from between the A and B chains, to be visualized.
Ligand-induced conformational change in penicillin acylase
Journal of Molecular Biology, 1998
The enzyme penicillin acylase (penicillin amidohydrolase EC 3.5.1.11) catalyses the cleavage of the amide bond in the benzylpenicillin (penicillin G) side-chain to produce phenylacetic acid and 6-aminopenicillanic acid (6-APA). The enzyme is of great pharmaceutical importance, as the product 6-APA is the starting point for the synthesis of many semi-synthetic penicillin antibiotics. Studies have shown that the enzyme is speci®c for hydrolysis of phenylacetamide derivatives, but is more tolerant of features in the rest of the substrate. It is this property that has led to many other applications for the enzyme, and greater knowledge of the enzyme's structure and speci®city could facilitate engineering of the enzyme, enhancing its potential for chemical and industrial applications.
Protein Engineering Design and Selection, 2004
Penicillin acylase catalyses the condensation of Casubstituted phenylacetic acids with b-lactam nucleophiles, producing semi-synthetic b-lactam antibiotics. For efficient synthesis a low affinity for phenylacetic acid and a high affinity for Ca-substituted phenylacetic acid derivatives is desirable. We made three active site mutants, aF146Y, bF24A and aF146Y/bF24A, which all had a 2-to 10-fold higher affinity for Ca-substituted compounds than wildtype enzyme. In addition, bF24A had a 20-fold reduced affinity for phenylacetic acid. The molecular basis of the improved properties was investigated by X-ray crystallography. These studies showed that the higher affinity of aF146Y for (R)-a-methylphenylacetic acid can be explained by van der Waals interactions between aY146:OH and the Ca-substituent. The bF24A mutation causes an opening of the phenylacetic acid binding site. Only (R)-a-methylphenylacetic acid, but not phenylacetic acid, induces a conformation with the ligand tightly bound, explaining the weak binding of phenylacetic acid. A comparison of the bF24A structure with other open conformations of penicillin acylase showed that bF24 has a fixed position, whereas aF146 acts as a flexible lid on the binding site and reorients its position to achieve optimal substrate binding.
The Refined Crystallographic Structure of aDD-Peptidase Penicillin-target Enzyme at 1.6 Å Resolution
Journal of Molecular Biology, 1995
The D-alanyl-D-alanine peptidase from Streptomyces sp. R61 is a 37,500 Cell Biology and Institute of dalton exocellular enzyme that has served as a model for membrane-bound peptidases that are involved in bacterial cell wall biosynthesis. Inhibition Materials Science, University of Connecticut, Storrs of these enzymes by b-lactam antibiotics ultimately leads to bacterial cell death. The X-ray crystal structure of the R61 D-alanyl-D-alanine peptidase CT 06269-3125, USA has been solved using multiple isomorphous replacement, simulated annealing and least squares refinement. The space group and unit cell parameters are P2 1 2 1 2 1 with a = 51.1 Å, b = 67.3 Å and c = 102.4 Å. The structure has been refined using 2s data to 1.6 Å resolution with a crystallographic R-factor of 0.148. The model contains 347 residues (2938 atoms) and 254 solvent molecules. The overall temperature factor is 9.6 Å 2 , and the estimated coordinate error is 0.14 Å. The protein consists of a single polypeptide chain organized into two regions. One region contains a nine-stranded antiparallel b-sheet with helices on both faces; this region includes both the amino and carboxyl termini. The second region is all helical. Sixty percent of the residues occur in helices or b-sheet. The reactive Ser62 is found between the two regions of the enzyme at the amino end of the protein's longest helix which begins with one turn of 3 10 helix and continues with four turns of a-helix. The active site is an elongated pocket that contains four basic and four aromatic residues. An oxyanion hole is formed by Ser62 NH and Thr301 NH. The pocket also contains the few key residues that are conserved in all penicillin-binding proteins and b-lactamases. Two of these residues, Lys65 and Tyr159, are among the 16 side-chains that take on multiple conformations in the R61 crystal structure. Three of the 12 proline rings adopt two conformations which we believe has not been previously reported. There is no anionic acid equivalent to the catalytic Glu166 found in Class A b-lactamases. Two ordered water molecules (O507 and O644) are found buried in the active site and hydrogen-bonded to each other (2.6 Å). O507 could potentially act as the hydrolytic water molecule for deacylation.
Crystal structures of two domains of bifunctional enzyme: human PAPS synthetase
Acta Crystallographica Section A, 2005
Glutamylcysteine synthetase (GCS) catalyzes the first and ratelimiting step of biosynthesis of a ubiquitous tripeptide glutathione and is a target for development of potential therapeutic agents against parasites and cancer. L-Buthionine-(SR)-sulfoximine (BSO) is a wellknown potent inhibitor of GCS. Clinical trials of BSO have been carried out against alkylating or platinating agent resistance cancers. Crystallographic analyses of GCS-BSO complex will provide an important clue to the catalytic mechanism and structure-assisted drug design for any species of GCSs. The crystal of E. coli GCS in complex with BSO belongs to the space group P2 1 with unit cell constants of a=70.5 Å, b=97.6 Å, c=102.7 Å and =109.5°. The current model was refined to an Rfactor of 21% (R free =24%). g-Phosphate of ATP has already been transferred to the NS sulfoximine nitrogen atom of BSO. We have shown that the cysteine-binding site of the GCS is inductively formed at the binding of cysteine substrate with turn of side chains of Tyr-241 and Tyr-300 to make hydrogen bonds with the carboxyl group of cysteine that w-carboxyl group of BSO mimics. The binding of BSO to the enzyme induces the turn of the side chain of Tyr-241 in spite of the lack of BSO's w-carboxyl group. This conformational change of the side chain may be stabilized by van der Waals interaction between the side chain of Tyr-241 and the glutamate moiety in BSO.
Biochemistry, 2010
O-Acetylserine sulfhydrylase is a pyridoxal 5 0 -phosphate (PLP)-dependent enzyme that catalyzes the final step in the cysteine biosynthetic pathway in enteric bacteria and plants, the replacement of the βacetoxy group of O-acetyl-L-serine (OAS) by a thiol to give L-cysteine. Previous studies of the K41A mutant enzyme showed L-methionine bound in an external Schiff base (ESB) linkage to PLP as the enzyme was isolated. The mutant enzyme exists in a closed form, optimizing the orientation of the cofactor PLP and properly positioning active site functional groups for reaction. The trigger for closing the active site upon formation of the ESB is thought to be interaction of the substrate R-carboxylate with the substrate-binding loop comprised of T68, S69, G70, and N71, and Q142, which is positioned above the cofactor as one looks into the active site. To probe the contribution of these residues to the active site closing and orientation of PLP in the ESB, T68, S69, N71, and Q142 were changed to alanine. Absorbance, fluorescence, near UV-visible CD, and 31 P NMR spectral studies and pre-steady state kinetic studies were used to characterize the mutant enzymes. All of the mutations affect closure of the active site, but to differing extents. In addition, the site appears to be more hydrophilic given that the ESBs do not exhibit a significant amount of the enolimine tautomer. No buildup of the R-aminoacrylate intermediate (AA) is observed for the T68A and Q142A mutant enzymes. However, pyruvate is produced at a rate much greater than that of the wild-type enzyme. Data suggest that T68 and Q142 play a role in stabilizing the AA. Both residues donate a hydrogen bond to one of the carboxylate oxygens of the methionine ESB and may also be responsible for the proper orientation of the ESB to generate the AA. The S69A and N71A mutants exhibit formation of the AA, but the rate constant for its formation from the ESB is decreased by 1 order of magnitude compared to that of the wild type. S69 donates a hydrogen bond to the substrate carboxylate in the ESB, while N71 donates hydrogen bonds to O3 0 of the cofactor and the carboxylate of the ESB; these side chains may also affect the orientation of the ESB. Data suggest that interaction of intermediates with the substrate-binding loop and Q142 gives a properly aligned Michaelis complex and facilitates the β-elimination reaction.
Trapping of an Acyl–Enzyme Intermediate in a Penicillin-binding Protein (PBP)-catalyzed Reaction
Journal of Molecular Biology, 2008
Class A penicillin-binding proteins (PBPs) catalyze the last two steps in the biosynthesis of peptidoglycan, a key component of the bacterial cell wall. Both reactions, glycosyl transfer (polymerization of glycan chains) and transpeptidation (cross-linking of stem peptides), are essential for peptidoglycan stability and for the cell division process, but remain poorly understood. The PBP-catalyzed transpeptidation reaction is the target of β-lactam antibiotics, but their vast employment worldwide has prompted the appearance of highly resistant strains, thus requiring concerted efforts towards an understanding of the transpeptidation reaction with the goal of developing better antibacterials. This goal, however, has been elusive, since PBP substrates are rapidly deacylated. In this work, we provide a structural snapshot of a "trapped" covalent intermediate of the reaction between a class A PBP with a pseudo-substrate, N-benzoyl-D-alanylmercaptoacetic acid thioester, which partly mimics the stem peptides contained within the natural, membrane-associated substrate, lipid II. The structure reveals that the D-alanyl moiety of the covalent intermediate (N-benzoyl-D-alanine) is stabilized in the cleft by a network of hydrogen bonds that place the carbonyl group in close proximity to the oxyanion hole, thus mimicking the spatial arrangement of β-lactam antibiotics within the PBP active site. This arrangement allows the target bond to be in optimal position for attack by the acceptor peptide and is similar to the structural disposition of β-lactam antibiotics with PBP clefts. This information yields a better understanding of PBP catalysis and could provide key insights into the design of novel PBP inhibitors.