Arg292----Val] or [Arg292----Leu] mutation enhances the reactivity of Escherichia coli aspartate aminotransferase with aromatic amino acids

Arg292 of E. coli aspartate aminotransferase was substituted with valine or leucine by site-directed mutagenesis. In comparison with the wild-type enzyme, either of the mutant enzymes showed a decrease by over 5 orders of magnitude of kcat/km values for aspartate and glutamate. This supports the contention that Arg292 is important for determining the specificity of this enzyme for dicarboxylic substrates. In contrast, mutant enzymes displayed a 5- to 10-fold increase in kcat/Km values for aromatic amino acids as substrates. Thus, introduction of an uncharged, hydrophobic side chain into position 292 leads to a striking alteration in substrate specificity of this enzyme, thereby improving catalytic efficiency toward aromatic amino acids.     

Crystal structures of branched-chain amino acid aminotransferase complexed with glutamate and glutarate: true reaction intermediate and double substrate recognition of the enzyme

Branched-chain amino acid aminotransferase (BCAT), which has pyridoxal 5'-phosphate as a cofactor, is a key enzyme in the biosynthetic pathway of hydrophobic amino acids (leucine, isoleucine, and valine). The enzyme reversibly catalyzes the transfer of the amino group of a hydrophobic amino acid to 2-oxoglutarate to form a 2-oxo acid and glutamate. Therefore, the active site of BCAT should have a mechanism to enable recognition of an acidic amino acid as well as a hydrophobic amino acid (double substrate recognition). The three-dimensional structures of Escherichia coli BCAT (eBCAT) in complex with the acidic substrate (glutamate) and the acidic substrate analogue (glutarate) have been determined by X-ray diffraction at 1.82 and 2.15 A resolution, respectively. The enzyme is a homo hexamer, with the polypeptide chain of the subunit folded into small and large domains, and an interdomain loop. The eBCAT in complex with the natural substrate, glutamate, was assigned as a ketimine as the most probable form based upon absorption spectra of the crystal complex and the shape of the residual electron density corresponding to the cofactor-glutamate bond structure. Upon binding of an acidic substrate, the interdomain loop approaches the substrate to shield it from the solvent region, as observed in the complex with a hydrophobic substrate. Both the acidic and the hydrophobic side chains of the substrates are bound to almost the same position in the pocket of the enzyme and are identical in structure. The inner side of the pocket is mostly hydrophobic to accommodate the hydrophobic side chain but has four sites to coordinate with the gamma-carboxylate of glutamate. The mechanism for the double substrate recognition observed in eBCAT is in contrast to those in aromatic amino acid and histidinol-phosphate aminotransferases. In an aromatic amino acid aminotransferase, the acidic side chain is located at the same position as that for the aromatic side chain because of large-scale rearrangements of the hydrogen bond network. In the histidinol-phosphate aminotransferase, the acidic and basic side chains are located at different sites and interact with different residues of the disordered loop.     

Dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia exhibits activity against a substrate containing a 4-hydroxyproline residue

The crystal structure of dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia was determined at 2.8-A resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space group P4(3)2(1)2, with unit cell parameters a = b = 105.9 A and c = 161.9 A. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal beta-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes. Stenotrophomonas dipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In the Stenotrophomonas enzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.     

The function of hydrophobic residues in the catalytic cleft of Streptococcus pneumoniae hyaluronate lyase. Kinetic characterization of mutant enzyme forms

Streptococcus pneumoniae hyaluronate lyase is a surface antigen of this Gram-positive human bacterial pathogen. The primary function of this enzyme is the degradation of hyaluronan, which is a major component of the extracellular matrix of the tissues of vertebrates and of some bacteria. The enzyme degrades its substrate through a beta-elimination process called proton acceptance and donation. The inherent part of this degradation is a processive mode of action of the enzyme degrading hyaluronan into unsaturated disaccharide hyaluronic acid blocks from the reducing to the nonreducing end of the polymer following the initial random endolytic binding to the substrate. The final degradation product is the unsaturated disaccharide hyaluronic acid. The residues of the enzyme that are involved in various aspects of such degradation were identified based on the three-dimensional structures of the native enzyme and its complexes with hyaluronan substrates of various lengths. The catalytic residues were identified to be Asn(349), His(399), and Tyr(408). The residues responsible for the release of the product of the reaction were identified as Glu(388), Asp(398), and Thr(400), and they were termed negative patch. The hydrophobic residues Trp(291), Trp(292), and Phe(343) were found to be responsible for the precise positioning of the substrate for enzyme catalysis and named hydrophobic patch. The comparison of the specific activities and kinetic properties of the wild type and the mutant enzymes involving the hydrophobic patch residues W292A, F343V, W291A/W292A, W292A/F343V, and W291A/W292A/F343V allowed for the characterization of every mutant and for the correlation of the activity and kinetic properties of the enzyme with its structure as well as the mechanism of catalysis.     

Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin

To test the role of Asp-189 which is located at the base of the substrate binding pocket in determining the specificity of trypsin toward basic substrates, this residue was replaced with a lysine residue by site-directed mutagenesis. Both rat trypsinogen and Lys-189 trypsinogen were expressed and secreted into the periplasmic space of Escherichia coli. The proteins were purified to homogeneity and activated by porcine enterokinase, and their catalytic activities were determined on natural and synthetic substrates. Lys-189 trypsin displayed no catalytic activity toward arginyl and lysyl substrates. Further, there was no compensatory change in specificity toward acidic substrates; no cleavage of aspartyl or glutamyl bonds was detected. Additional studies of substrate specificity involving gas-phase sequence analyses of digested natural substrates revealed an inherent but low chymotrypsin-like activity of trypsin. This activity was retained but modified by the Asp to Lys change at position 189. In addition to hydrolyzing phenylalanyl and tyrosyl peptide bonds, the mutant enzyme has the unique property of cleaving leucyl bonds. On the basis of computer graphic modeling studies of the Lys-189 side chain, it appears that the positively charged NH2 group is directed outside the substrate binding pocket. The resulting hydrophobic cavity may explain the altered substrate specificity of the mutant enzyme. The relatively low chymotrypsin-like activity of both recombinant enzymes may be due to distorted positioning of the scissile bond with respect to the catalytic triad rather than to the lack of sufficient interaction between the hydrophobic side chains and the substrate binding pocket of the enzyme.     

Structural Basis for the Recognition of Mycolic Acid Precursors by KasA,a Condensing Enzyme and Drug Target from Mycobacterium Tuberculosis

The survival of Mycobacterium tuberculosis depends on mycolic acids, very long α-alkyl-β-hydroxy fatty acids comprising 60–90 carbon atoms. However, despite considerable efforts, little is known about how enzymes involved in mycolic acid biosynthesis recognize and bind their hydrophobic fatty acyl substrates. The condensing enzyme KasA is pivotal for the synthesis of very long (C_38–42) fatty acids, the precursors of mycolic acids. To probe the mechanism of substrate and inhibitor recognition by KasA, we determined the structure of this protein in complex with a mycobacterial phospholipid and with several thiolactomycin derivatives that were designed as substrate analogs. Our structures provide consecutive snapshots along the reaction coordinate for the enzyme-catalyzed reaction and support an induced fit mechanism in which a wide cavity is established through the concerted opening of three gatekeeping residues and several α-helices. The stepwise characterization of the binding process provides mechanistic insights into the induced fit recognition in this system and serves as an excellent foundation for the development of high affinity KasA inhibitors.     

The substrate activation process in the catalytic reaction of Escherichia coli aromatic amino acid aminotransferase

Aromatic amino acid aminotransferase is active toward both aromatic and dicarboxylic amino acids, and the mechanism for this dual substrate recognition has been an issue in the enzymology of this enzyme. Here we show that, in the reactions with aromatic and dicarboxylic ligands, the pK(a) of the Schiff base formed between the coenzyme pyridoxal 5'-phosphate and Lys258 or the substrate increases successively from 6.6 in the unliganded enzyme to approximately 8.8 in the Michaelis complex and to >10.5 in the external Schiff base complex. Mutations of Arg292 and Arg386 to Leu, which mimic neutralization of the positive charges of the two arginine residues by the ligand carboxylate groups, increased the Schiff base pK(a) by 0.1 and 0.7 unit, respectively. In contrast to these moderate effects of the Arg mutations, the cleavage of the Lys258 side chain of the Schiff base, which was brought about by preparing a mutant enzyme in which Lys258 was changed to Ala and the Schiff base was reconstituted with methylamine, produced the Schiff base pK(a) value of 10.2, that being 3.6 units higher than that of the wild-type enzyme. The observation indicates that the Schiff base pK(a) in the enzyme is lowered by the torsion around the C4-C4' axis of the Schiff base and suggests that the pK(a) is mainly controlled by changing the torsion angle during the course of catalysis. This mechanism, first observed for the reaction of aspartate aminotransferase with aspartate [Hayashi, H., Mizuguchi, H., and Kagamiyama, H. (1998) Biochemistry 37, 15076-15085], does not require the electrostatic contribution from the omega-carboxylate group of the substrate, and can explain why in aromatic amino acid aminotransferase the aromatic substrates can increase the Schiff base pK(a) during catalysis to the same extent as the dicarboxylic substrates. This is the first example in which the torsion pK(a) coupling of the pyridoxal 5'-phosphate Schiff base has been demonstrated in pyridoxal enzymes other than aspartate aminotransferase, and suggests the generality of the mechanism in the catalysis of aminotransferases related to aspartate aminotransferase.     

Redesigning the substrate specificity of omega-aminotransferase for the kinetic resolution of aliphatic chiral amines

Substrate specificity of the omega-aminotransferase obtained from Vibrio fluvialis (omega-ATVf) was rationally redesigned for the kinetic resolution of aliphatic chiral amines. omega-ATVf showed unique substrate specificity toward aromatic amines with a high enantioselectivity (E > 100) for (S)-enantiomers. However, the substrate specificity of this enzyme was much narrower toward aliphatic amines. To overcome the narrow substrate specificity toward aliphatic amines, we redesigned the substrate specificity of omega-ATVf using homology modeling and the substrate structure- activity relationship. The homology model and the substrate structure-activity relationship showed that the active site of omega-ATVf consists of one large substrate-binding site and another small substrate-binding site. The key determinant in the small substrate-binding site was D25, whose role was expected to mask R415 and to generate the electrostatic repulsion with the substrate's alpha-carboxylate group. In the large substrate-binding site, R256 was predicted to recognize the alpha-carboxylate group of substrate thus obeying the dual substrate recognition mechanism of aminotransferase subgroup II enzymes. Among the several amino acid residues in the large substrate-binding site, W57 and W147, with their bulky side chains, were expected to restrict the recognition of aliphatic amines. Two mutant enzymes, W57G and W147G, showed significant changes in their substrate specificity such that they catalyzed transamination of a broad range of aliphatic amines without losing the original activities toward aromatic amines and enantioselectivity.     

Substrate recognition and selectivity of peptide deformylase. Similarities and differences with metzincins and thermolysin.

The substrate specificity of Escherichia coli peptide deformylase was investigated by measuring the efficiency of the enzyme to cleave formyl- peptides of the general formula Fo-Xaa-Yaa-NH2, where Xaa represents a set of 27 natural and unusual amino acids and Yaa corresponds to a set of 19 natural amino acids. Substrates with bulky hydrophobic side-chains at the P1' position were the most efficiently cleaved, with catalytic efficiencies greater by two to five orders of magnitude than those associated with polar or charged amino acid side-chains. Among hydrophobic side-chains, linear alkyl groups were preferred at the P1' position, as compared to aryl-alkyl side-chains. Interestingly, in the linear alkyl substituent series, with the exception of norleucine, deformylase exhibits a preference for the substrate containing Met in the P1' position. Next, the influence in catalysis of the second side-chain was studied after synthesis of 20 compounds of the formula Fo-Nle-Yaa-NH2. Their deformylation rates varied within a range of only one order of magnitude. A 3D model of the interaction of PDF with an inhibitor was then constructed and revealed indeed the occurrence of a deep and hydrophobic S1' pocket as well as the absence of a true S2' pocket. These analyses pointed out a set of possible interactions between deformylase and its substrates, which could be the ground driving substrate specificity. The validity of this enzyme:substrate docking was further probed with the help of a set of site-directed variants of the enzyme. From this, the importance of residues at the bottom of the S1' pocket (Ile128 and Leu125) as well as the hydrogen bond network that the main chain of the substrate makes with the enzyme were revealed. Based on the numerous homologies that deformylase displays with thermolysin and metzincins, a mechanism of enzyme:substrate recognition and hydrolysis could finally be proposed. Specific features of PDF with respect to other members of the enzymes with motif HEXXH are discussed.     

Structural features of glutamine substrates for transglutaminases. Specificities of human plasma factor XIIIa and the guinea pig liver enzyme toward synthetic peptides

Studies on the glutamine substrate specificities of human plasma factor XIIIa and guinea pig liver transglutaminase have been made using variants of the synthetic peptide substrate, Ser-Val-Leu-Ser-Leu-Ser-Gln-Ser-Lys-Val-Leu-Pro-Val-Pro-Glu. The sequence of this effective peptide substrate corresponds to the primary site of factor XIIIa-catalyzed amine incorporation into beta-casein, the most sensitive known macromolecular substrate for this enzyme (Gorman, J.J., and Folk, J.E. (1980) J. Biol. Chem. 255, 419-427). Variations in specificity observed with factor XIIIa for peptides containing single substitutions and multiple substitutions in this sequence are indications that several important determinants for enzyme recognition are contained therein. Among these are several of the hydrophobic amino acid residues and the lysine residue. Less pronounced changes in specificity occur with the liver enzyme and the differences in effects of the various substitutions reveal important differences in specificity requirements of factor XIIIa and the liver enzyme. Comparisons of the activities of the enzymes toward the synthetic peptides to their activities toward macromolecular substrates suggest that higher order macromolecular structural features contribute to specificity.     

Display of active subtilisin 309 on phage: analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors

Many attempts have been made to endow enzymes with new catalytic activities. One general strategy involves the creation of random combinatorial libraries of mutants associated with an efficient screening or selection scheme. Phage display has been shown to greatly facilitate the selection of polypeptides with desired properties by establishing a close link between the polypeptide and the gene that encodes it. Selection of phage displayed enzymes for new catalytic activities remains a challenge. The aim of this study was to display the serine protease subtilisin 309 (savinase) from Bacillus lentus on the surface of filamentous fd phage and to develop selection schemes that allow the extraction of subtilisin variants with a changed substrate specificity from libraries. Subtilisins are produced as secreted preproenzyme that mature in active enzyme autocatalytically. They have a broad substrate specificity but exhibit a significant preference for hydrophobic residues and very limited reactivity toward charged residues at the P4 site in the substrate. Here, we show that savinase can be functionally displayed on phage in the presence of the proteic inhibitor CI2. The free enzyme is released from its complex with CI2 upon addition of the anionic detergent LAS. The phage-enzyme can be panned on streptavidin beads after labelling by reaction with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-l-Ala-l-Ala-l-P ro-Phe(P)-diphenyl ester. Reactions of libraries, in which residues 104 and 107 forming part of the S4 pocket have been randomised, with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-alpha-l-Lys-l-A la-l-Pro-Phe(P)-diphenylester allowed us to select enzymes with increased specific activity for a substrate containing a lysine in P4. Parameters influencing the selection as for instance the efficiency of maturation of mutant enzymes in libraries have been investigated.     

Crystal structure of a thermophilic alcohol dehydrogenase substrate complex suggests determinants of substrate specificity and thermostability

The crystal structure of a thermophilic alcohol dehydrogenase (TBAD) from Thermoanaerobacter brockii has been determined in a binary complex with sec-butanol as substrate to a resolution of 3.0 A. Van der Waals interactions of the carbon C1 atom of sec-butanol with atoms in His59, Ala85, Trp110, Asp150, and Leu294 account for the substrate preference of this enzyme for secondary over primary alcohols. A crevice from the surface to the active site provides access for substrates and products. This opening is lined with the hydrophobic residues Ile49, Leu107, Trp110, Tyr267, Leu294 as well as Cys283 and Met285 from another molecule within the tetrameric assembly. This might explain the tolerance of this enzyme toward organic solvents. The zinc ion occupies a position in the active site, which is too remote for direct interaction with the alcohol group. A mechanism is suggested whereby the introduction of NADP would trigger a displacement of the zinc ion to its catalytic site. Features important for the unusually high melting temperature of 98 degrees C are suggested by comparison to the crystal structure of a highly homologous mesophilic alcohol dehydrogenase from Clostridium beijerinckii (CBAD). The thermophilic enzyme has a more hydrophilic exterior, a more hydrophobic interior, a smaller surface area, more prolines, alanines, and fewer serines than CBAD. Furthermore, in the thermophilic enzyme the number of all types of intersubunit interactions in these tetrameric enzymes is increased: more salt bridges, hydrogen bonds, and hydrophobic interactions. All these effects combined can account for the higher melting temperature of the thermophilic enzyme.     

The structure of rhamnose isomerase from Escherichia coli and its relation with xylose isomerase illustrates a change between inter and intra-subunit complementation during evolution

Using a new expression construct, rhamnose isomerase from Escherichia coli was purified and crystallized. The crystal structure was solved by multiple isomorphous replacement and refined to a crystallographic residual of 17.4 % at 1.6 A resolution. Rhamnose isomerase is a tight tetramer of four (beta/alpha)(8)-barrels. A comparison with other known structures reveals that rhamnose isomerase is most similar to xylose isomerase. Alignment of the sequences of the two enzymes based on their structures reveals a hitherto undetected sequence identity of 13 %, suggesting that the two enzymes evolved from a common precursor. The structure and arrangement of the (beta/alpha)(8)-barrels of rhamnose isomerase are very similar to xylose isomerase. Each enzyme does, however, have additional alpha-helical domains, which are involved in tetramer association, and largely differ in structure. The structures of complexes of rhamnose isomerase with the inhibitor l-rhamnitol and the natural substrate l-rhamnose were determined and suggest that an extended loop, which is disordered in the native enzyme, becomes ordered on substrate binding, and may exclude bulk solvent during catalysis. Unlike xylose isomerase, this loop does not extend across a subunit interface but contributes to the active site of its own subunit. It illustrates how an interconversion between inter and intra-subunit complementation can occur during evolution. In the crystal structure (although not necessarily in vivo) rhamnose isomerase appears to bind Zn(2+) at a "structural" site. In the presence of substrate the enzyme also binds Mn(2+) at a nearby "catalytic" site. An array of hydrophobic residues, not present in xylose isomerase, is likely to be responsible for the recognition of l-rhamnose as a substrate. The available structural data suggest that a metal-mediated hydride-shift mechanism, which is generally favored for xylose isomerase, is also feasible for rhamnose isomerase.     

Trapping conformational states along ligand-binding dynamics of peptide deformylase: the impact of induced fit on enzyme catalysis

For several decades, molecular recognition has been considered one of the most fundamental processes in biochemistry. For enzymes, substrate binding is often coupled to conformational changes that alter the local environment of the active site to align the reactive groups for efficient catalysis and to reach the transition state. Adaptive substrate recognition is a well-known concept; however, it has been poorly characterized at a structural level because of its dynamic nature. Here, we provide a detailed mechanism for an induced-fit process at atomic resolution. We take advantage of a slow, tight binding inhibitor-enzyme system, actinonin-peptide deformylase. Crystal structures of the initial open state and final closed state were solved, as well as those of several intermediate mimics captured during the process. Ligand-induced reshaping of a hydrophobic pocket drives closure of the active site, which is finally "zipped up" by additional binding interactions. Together with biochemical analyses, these data allow a coherent reconstruction of the sequence of events leading from the encounter complex to the key-lock binding state of the enzyme. A "movie" that reconstructs this entire process can be further extrapolated to catalysis.     

Interconversion of ketoprofen recognition in firefly luciferase-catalyzed enantioselective thioesterification reaction using from Pylocoeria miyako (PmL) and Hotaria parvura (HpL) just by mutating two amino acid residues

We identified the critical amino acid residues for substrate recognition using two firefly luciferases from Pylocoeria miyako (PmL) and Hotaria parvura (HpL), as these two luciferase enzymes exhibit different activities toward ketoprofen. Specifically, PmL can catalyze the apparent enantioselective thioesterification reaction, while HpL cannot. By comparing the amino acid sequences around the active site, we identified two residues (I350 and M397 in PmL and F351 and S398 in HpL) that were different between the two enzymes, and the replacement of these amino acids resulted in changing the ketoprofen recognition pattern. The inactive HpL was converted to the active enzyme toward ketoprofen and vice versa for PmL. These residues also affected the enantioselectivity toward ketoprofen; however, the bioluminescent color was not affected. In addition, using molecular dynamics calculations, the replacement of these two amino acids induced changes in the state of hydrogen bonding between ketoprofen and the S349 side chain through the active site water. As S349 is not considered to influence color tuning, these changes specifically caused the differences in ketoprofen recognition in the enzyme.     

Replacement of an interdomain residue Val39 of Escherichia coli aspartate aminotransferase affects the catalytic competence without altering the substrate specificity of the enzyme

Three mutant Escherichia coli aspartate aminotransferases in which Val39 was changed to Ala, Leu, and Phe by site-directed mutagenesis were prepared and characterized. Among the three mutant and the wild-type enzymes, the Leu39 enzyme had the lowest Km values for dicarboxylic substrates. The Km values of the Ala39 enzyme for dicarboxylates were essentially the same as those of the wild-type (Val39) enzyme. These two mutant enzymes showed essentially the same kcat values for dicarboxylic substrates as did the wild-type enzyme. On the other hand, incorporation of a bulky side-chain at position 39 (Phe39 enzyme) decreased both the affinity (1/Km) and catalytic ability (kcat) toward dicarboxylic substrates. These results show that the position 39 residue is involved in the modulation of both the binding of dicarboxylic substrates to enzyme and the catalytic ability of the enzyme. Although the replacement of Val39 with other residues altered both the kcat and Km values toward various substrates including dicarboxylic and aromatic amino acids and the corresponding oxo acids, it did not alter the ratio of the kcat/Km value of the enzyme toward a dicarboxylic substrate to that for an aromatic substrate. The affinity for aromatic substrates was not affected by changing the residue at position 39. These data indicate that, although the side chain bulkiness of the residue at position 39 correlates well with the activity toward aromatic substrates in the sequence alignment of several aminotransferases [Seville, M., Vincent M.G., & Hahn, K. (1988) Biochemistry 27, 8344-8349], the residue does not seem to be involved in the recognition of aromatic substrates.     

Thermodynamic and mutational studies of l-N-carbamoylase from Sinorhizobium meliloti CECT 4114 catalytic centre

Purified site-directed mutants of Sinorhizobium meliloti CECT 4114 l-N-carbamoylase (SmLcar) in which Glu132, His230, Asn279 and Arg292 were replaced have been studied by kinetic methods and isothermal titration calorimetry (ITC). The importance of His230, Asn279 and Arg292 residues in the recognition of N-carbamoyl-l-alpha-amino acids has been proved. The role of Glu132 has been confirmed in substrate hydrolysis. ITC has confirmed two Ni atoms per monomer of wild type enzyme, and two equal and independent substrate binding sites (one per monomer). Homology modelling of SmLcar supports the importance of His87, His194, His386, Glu133 and Asp98 in metal binding. A comprehensive reaction mechanism is proposed on the basis of binding experiments measured by ITC, kinetic assays, and homology of the active centre with beta-alanine synthase from Saccharomyces kluyveri and other enzymes.     

Recognition of acetylated proteins: lessons from an ancient family of enzymes

The structure of a Sir2-like enzyme in complex with an acetylated peptide substrate has been solved, and provides the first glimpse into the mechanism of substrate recognition by this highly conserved family of enzymes.     

Poly-alpha-glutamic acid synthesis using a novel catalytic activity of RimK from Escherichia coli K-12

Poly-L-α-amino acids have various applications because of their biodegradable properties and biocompatibility. Microorganisms contain several enzymes that catalyze the polymerization of L-amino acids in an ATP-dependent manner, but the products from these reactions contain amide linkages at the side residues of amino acids: e.g., poly-γ-glutamic acid, poly-ε-lysine, and cyanophycin. In this study, we found a novel catalytic activity of RimK, a ribosomal protein S6-modifying enzyme derived from Escherichia coli K-12. This enzyme catalyzed poly-α-glutamic acid synthesis from unprotected L-glutamic acid (Glu) by hydrolyzing ATP to ADP and phosphate. RimK synthesized poly-α-glutamic acid of various lengths; matrix-assisted laser desorption ionization-time of flight-mass spectrometry showed that a 46-mer of Glu (maximum length) was synthesized at pH 9. Interestingly, the lengths of polymers changed with changing pH. RimK also exhibited 86% activity after incubation at 55°C for 15 min, thus showing thermal stability. Furthermore, peptide elongation seemed to be catalyzed at the C terminus in a stepwise manner. Although RimK showed strict substrate specificity toward Glu, it also used, to a small extent, other amino acids as C-terminal substrates and synthesized heteropeptides. In addition, RimK-catalyzed modification of ribosomal protein S6 was confirmed. The number of Glu residues added to the protein varied with pH and was largest at pH 9.5.     

Identification of the principal catalytically important acidic residue of 3-hydroxy-3-methylglutaryl coenzyme A reductase

Kinetic analysis of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase has implicated a glutamate or aspartate residue in (i) formation of mevaldate thiohemiacetal by proton transfer to the carbonyl oxygen of mevaldate and (ii) enhanced ionization of CoASH by the resulting enzyme carboxylate anion, facilitating attack by CoAS- on the carbonyl carbon of mevaldate (Veloso, D., Cleland, W. W., and Porter, J. W. (1981) Biochemistry 81, 887-894). Although neither the identity of this acidic residue nor its location is known, the catalytic domains of 11 sequenced HMG-CoA reductases contain only 3 conserved acidic residues. For HMG-CoA reductase of Pseudomonas mevalonii, these residues are Glu52, Glu83, and Asp183. To identify the acidic residue that functions in catalysis, we generated mutants having alterations in these residues. The mutant proteins were expressed, purified, and characterized. Mutational alteration of residues Glu52 or Asp183 of P. mevalonii HMG-CoA reductase yielded enzymes with significant, but in some cases reduced, activity (Vmax = 100% Asp183----Ala, 65% Asp183----Asn, and 15% Glu52----Gln of wild-type activity, respectively). Although the activity of mutant enzymes Glu52----Gln and Asp183----Ala was undetectable under standard assay conditions, their Km values for substrates were 4-300-fold higher than those for wild-type enzyme. Km values for wild-type enzyme and for mutant enzymes Glu52----Gln and Asp183----Ala were, respectively: 0.41, 73, and 120 mM [R,S)-mevalonate); 0.080, 4.4, and 2.0 mM (coenzyme A); and 0.26, 4.4, and 1.0 mM (NAD+). By these criteria, neither Glu52 nor Asp183 is the acidic catalytic residue although each may function in substrate recognition. During chromatography on coenzyme A agarose or HMG-CoA agarose, mutant enzymes Asp183----Asn and Glu83----Gln behaved like wild-type enzyme. By contrast, and in support of a role for these residues in substrate recognition, mutant enzymes Glu52----Gln and Asp183----Ala exhibited impaired ability to bind to either support. Despite displaying Km values for substrates and chromatographic behavior on substrate affinity supports comparable to wild-type enzyme, only mutant enzyme Glu83----Gln was essentially inactive under all conditions studied (Vmax = 0.2% that of wild-type enzyme). Glutamate residue 83 of P. mevalonii HMG-CoA reductase, and consequently the glutamate of the consensus Pro-Met-Ala-Thr-Thr-Glu-Gly-Cys-Leu-Val-Ala motif of the catalytic domains of eukaryotic HMG-CoA reductases, is judged to be the acidic residue functional in catalysis.