The Effect of Hinge Mutations on Effector Binding and Domain Rotation in Escherichia coli D-3-Phosphoglycerate Dehydrogenase (original) (raw)
Related papers
The Journal of Biological Chemistry, 2007
D-3-Phosphoglycerate dehydrogenase (EC 1.1.1.95) from Escherichia coli contains two Gly-Gly sequences that have been shown previously to have the characteristics of hinge regions. One of these, Gly 336-Gly 337 , is found in the loop between the substrate binding domain and the regulatory domain. Changing these glycine residues to valine affected the sensitivity of the enzyme to inhibition by L-serine but not the extent of inhibition. The decrease in sensitivity was caused primarily by a decrease in the affinity of the enzyme for L-serine. These mutations also affected the domain rotation of the subunits in response to L-serine binding. A major conclusion of this study was that it defines a minimal limit on the necessary conformational changes leading to inhibition of enzyme activity. That is, some of the conformational differences seen in the native enzyme upon L-serine binding are not critical for inhibition, whereas others are maintained and may play important roles in inhibition and cooperativity. The structure of G336V demonstrates that the minimal effect of L-serine binding leading to inhibition of enzyme activity requires a domain rotation of approximately only 6°in just two of the four subunits of the enzyme that are oriented diagonally across from each other in the tetramer. Moreover the structures show that both pairs of Asn 190 to Asn 190 hydrogen bonds across the subunit interfaces are necessary for activity. These observations are consistent with the halfthe-sites activity, flip-flop mechanism proposed for this and other similar enzymes and suggest that the Asn 190 hydrogen bonds may function in the conformational transition between alternate half-the-site active forms of the enzyme.
Evolution of a new enzyme activity from the same motif fold
Molecular Microbiology, 2008
The host cell recognition protein of the Escherichia coli bacteriophage HK620 is a large homotrimeric tailspike that cleaves the O18A1 type O antigen. The crystal structure of HK620 tailspike determined in the apo and substrate-bound form is reported by Barbirz et al. in this issue of Molecular Microbiology. Lacking detectable sequence similarity, the fold and overall organization of the HK620 tailspike are similar to those of the tailspikes of the related phages P22 and Sf6. The substrate-binding site is intra-subunit in P22 and HK620 tailspikes, but inter-subunit in Sf6, demonstrating how phages can adapt the same protein fold to recognize different substrates. Keywords tailspike; evolution; polysaccharide; hydrolase; bacteriophage Conservation of structure in large oligomeric enzymes generally implies that function is conserved and the substrate-binding site is in a similar location, even in the absence of detectable sequence similarity. Two papers from the Seckler and Heinemann laboratories, one in this issue of Molecular Microbiology (Barbirz et al., 2008), refute this generality. Crystal structures of the 500+ residue, trimeric tailspikes from bacteriophages HK620 (Barbirz et al., 2008) and Sf6 (Müller et al., 2008) show that these enzymes, whose overall folds and organizations are conserved, differ markedly in their amino acid sequences, substrate specificities, enzymatic mechanisms, and locations of the substrate binding site. Barbirz et al. present a detailed characterization of the HK620 endo-N-acetylglucosamidase that degrades the O antigen lipopolysaccharide (LPS) of its Escherichia coli host. The crystal structures were determined to a remarkable 1.4 Å resolution for the apoenzyme and 1.6 Å when substrate-bound, allowing the first demonstration that the LPS of E. coli H TD2158 was indeed O18A1. Müller et al. (2008) provide a comparable analysis of the endorhamnosidase of the Shigella flexneri phage Sf6. These papers set a high standard for articles reporting an enzyme structure. Unlike laboratory strains of E. coli, the LPS of most environmental isolates is complete and is often further extended by a polymer of repeating carbohydrates called O antigen. Some bacteria also display a K antigen, a different capsular polysaccharide (PS) that was first defined as an antigen that masked O antigens. This distinction is no longer clear, as K and O antigens are now known to share more common features than differences (Schnaitman, 2001) Both consist of repeating units of two to seven, often modified, sugars that might be Contact E-mails.
Current Protein & Peptide Science, 2010
Coupling of structural flexibility and biological function is an essential feature of proteins. The role of relative domain movements in enzyme function has been evidenced in many cases. However, the way of communication between protein domains and its manifestation in their movements as well as in the biological function are rarely delineated. In this review we summarize comprehensive studies with a typical hinge-bending two-domain enzyme, 3-phosphoglycerate kinase. A possible mechanism is proposed by which the two substrates that bind to different domains trigger the operation of the molecular hinges, located in the interdomain region. Various crystal structures of the enzyme have been determined with different relative domain positions, suggesting that domain closure brings the two substrates together for the catalysis. Substrate-caused conformational changes in the binary and the ternary complexes have been tested with the solubilized enzyme using physical methods, such as differential scanning calorimetry, small angle X-ray scattering and infrared spectroscopy. The results indicated the existence of strong cooperativity between the two domains and that the presence of both substrates is necessary for the domain closure. Comparison of the atomic contacts in the structures has led to selection of conserved side-chains, which may be involved in the domain movement. On this basis a hypothesis was put forward about the molecular mechanism of interdomain co-operation. Enzyme kinetic, ligand binding and small angle X-ray scattering studies with various site-directed mutants have confirmed this hypothesis. Namely, a special H-bonding network (a double molecular switch) seems to be responsible for operation of the main molecular hinge at the beta-strand L under the concerted action of both substrates.
Part-of-the-sites binding and reactivity in the homooligomeric enzymes – facts and artifacts
Archives of Biochemistry and Biophysics, 2018
For a number of enzymes composed of several subunits with the same amino acid sequence, it was documented, or suggested, that binding of a ligand, or catalysis, is carried out by a single subunit. This phenomenon may be the result of a pre-existent asymmetry of subunits or a limiting case of the negative cooperativity, and is sometimes called "half-of-the-sites binding (or reactivity)" for dimers and could be called "part-of-the-sites binding (or reactivity)" for higher oligomers. In this article, we discuss molecular mechanisms that may result in "part-of-the-sites binding (and reactivity)", offer possible explanations why it may have a beneficial role in enzyme function, and point to experimental problems in documenting this behaviour. We describe some cases, for which such a mechanism was first reported and later disproved. We also give several examples of enzymes, for which this mechanism seems to be well documented, and profitable. A majority of enzymes identified in this study as half-of-the-sites binding (or reactive) use it in the flip-flop version, in which "half-of-the-sites" refers to a particular moment in time. In general, the various variants of the mechanism seems to be employed often by oligomeric enzymes for allosteric regulation to enhance the efficiency of enzymatic reactions in many key metabolic pathways.
Biochemistry, 1989
The allosteric transition of Escherichia coli aspartate transcarbamylase involves significant alterations in structure at both the quaternary and tertiary levels. On the tertiary level, the 240s loop (residues 230-245 of the catalytic chain) repositions, influencing the conformation of Arg-229, a residue near the aspartate binding site. In the T state, Arg-229 is bent out of the active site and may be stabilized in this position by an interaction with Glu-272. In the R state, the conformation of Arg-229 changes, allowing it to interact with the P-carboxylate of aspartate, and is stabilized in this position by a specific interaction with Glu-233. In order to ascertain the function of Arg-229, Glu-233, and Glu-272 in the catalytic and cooperative interactions of the enzyme, three mutant enzymes were created by site-specific mutagenesis. Arg-229 was replaced by Ala, while both Glu-233 and Glu-272 were replaced by Ser. The Arg-229-Ala and Glu-233-Ser enzymes exhibit 10 000-fold and 80-fold decreases in maximal activity, respectively, and they both exhibit a 2-fold increase in the aspartate concentration at half the maximal observed velocity, 'This work was supported by grants from the National Institutes of * To whom correspondence should be addressed. Health (DK1429 and GM26237). affinity and high catalytic activity has been proposed to occur in a concerted fashion based on kinetic, physicochemical, and structural analyses of both wild-type and mutant versions of the enzyme (Foote & Schachman, 1985; Krause et al., 1987; Abbreviations: holoenzyme, native enzyme composed of two catalytic trimers and three regulatory dimers; PALA, N-(phosphonoacetyl)-L-aspartate; Tris, tris(hydroxymethy1)aminomethane; T and R states, tense and relaxed conformations of the enzyme having low activity and affinity for substrates and high activity and affinity for substrates, respectively; [SI,,, substrate concentration at half the maximal observed specific activity; 80s loop, flexible loop of the enzyme comprising approximately amino acid residues 76-86 of the catalytic chain; 240s loop, flexible loop of the enzyme comprising approximately amino acid residues 230-245 of the catalytic chain.
Alternate-Site Enzyme Promiscuity
Angewandte Chemie International Edition, 2007
Enzyme promiscuity means, in the broadest terms, the ability of a given enzyme to catalyze distinctly different chemical transformations of natural or nonnatural substrates. Although it was originally thought to be a fairly rare event, research over the last few years has uncovered many more examples. It has also become clear that catalytic promiscuity has implications in evolutionary relationships. This intriguing frontier in enzymology has several important theoretical and practical facets. For example, the question of how proteins in nature evolve new functions such as antibiotic resistance or the ability to degrade man-made chemicals, both within months or years, is of fundamental significance and has been studied by applying the methods of directed evolution. [1c, 3] Moreover, the discovery of promiscuous behavior of wild-type (WT) enzymes or mutants thereof produced by protein engineering has the potential of expanding the repertoire of synthetic organic methodologies. In all studies reported so far, the promiscuous (secondary) reaction has been linked to the binding site of the reaction for which the enzyme is primarily known, generally involving some or all of the original catalytically active amino acids or metal centers. Examples are alkaline phosphatase catalyzed hydrolysis of p-nitrophenylsulfate, aminopeptidase-catalyzed hydrolysis of phosphoesters, lipase-catalyzed Michael additions of N-, [6] O-, [6] S-, [6] and C-nucleophiles, aldol additions, oligomerization of siloxanes, [9] racemasecatalyzed PLP-dependent aldol additions, and arylmalonate decarboxylase catalyzed aldol additions. Many of these studies involve protein engineering. To the best of our knowledge, no case of enzyme promiscuity has been reported in which the known natural catalytically active site is not involved. Herein we report the first example of this phenomenon, which we call alternate-site enzyme promiscuity.
Biochemistry, 2008
The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from the bacterium Pseudomonas aeruginosa is involved in the biosynthesis of several complex carbohydrates, including alginate, lipopolysaccharide, and rhamnolipid. Previous structural studies of this protein have shown that binding of substrates produces a rotation of the C-terminal domain, changing the active site from an open cleft in the apoenzyme into a deep, solvent inaccessible pocket where phosphoryl transfer takes place. We report herein site-directed mutagenesis, kinetic, and structural studies in examining the role of residues in the hinge between domains 3 and 4, as well as residues that participate in enzyme-substrate contacts and help form the multidomain "lid" of the active site. We find that the backbone flexibility of residues in the hinge region (e.g., mutation of proline to glycine/alanine) affects the efficiency of the reaction, decreasing k cat by ∼10-fold and increasing K m by ∼2-fold. Moreover, thermodynamic analyses show that these changes are due primarily to entropic effects, consistent with an increase in the flexibility of the polypeptide backbone leading to a decreased probability of forming a catalytically productive active site. These results for the hinge residues contrast with those for mutants in the active site of the enzyme, which have profound effects on enzyme kinetics (10 2 -10 3 -fold decrease in k cat /K m ) and also show substantial differences in their thermodynamic parameters relative to those of the wild-type (WT) enzyme. These studies support the concept that polypeptide flexibility in protein hinges may evolve to optimize and tune reaction rates.
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, 1996
Here we describe the three-dimensional structure of 4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp. strain CBS-3. This enzyme catalyzes the hydrolysis of 4-chlorobenzoyl-CoA to 4-hydroxybenzoyl-CoA. The molecular structure of the enzyme/4-hydroxybenzoyl-CoA complex was solved by the techniques of multiple isomorphous replacement, solvent flattening, and molecular averaging. Least-squares refinement of the protein model reduced the crystallographic R factor to 18.8% for all measured X-ray data from 30 to 1.8 Å resolution. The crystallographic investigation of this dehalogenase revealed that the enzyme is a trimer. Each subunit of the trimer folds into two distinct motifs. The larger, N-terminal domain is characterized by 10 strands of-pleated sheet that form two distinct layers which lie nearly perpendicular to one another. These layers of-sheet are flanked on either side by R-helices. The C-terminal domain extends away from the body of the molecule and is composed of three amphiphilic R-helices. This smaller domain is primarily involved in trimerization. The two domains of the subunit are linked together by a cation, most likely a calcium ion. The 4-hydroxybenzoyl-CoA molecule adopts a curved conformation within the active site such that the 4-hydroxybenzoyl and the adenosine moieties are buried while the pantothenate and pyrophosphate groups of the coenzyme are more solvent exposed. From the three-dimensional structure it is clear that Asp 145 provides the side-chain carboxylate group that adds to form the Meisenheimer intermediate and His 90 serves as the general base in the subsequent hydrolysis step. Many of the structural principles derived from this investigation may be directly applicable to other related enzymes such as crotonase. † This research was supported in part by grants from the NIH (GM36260 to D.D.M. and DK47814 to H.M.H.). ‡ Coordinates have been deposited in the Brookhaven Protein Data Bank under the identification code 1NZY.