Identification of two residues essential for the stringent substrate specificity and active site stability of the prokaryotic l-arginine:glycine amidinotransferase CyrA

A novel prokaryotic l-arginine:glycine amidinotransferase (CyrA; EC2.1.4.1) is involved in the biosynthesis of the polyketide-derived cytotoxin cylindrospermopsin in the cyanobacterium Cylindrospermopsis raciborskii AWT250, and was previously characterized with regard to kinetic mechanism and substrate specificity [Muenchhoff J et al. (2010) FEBS J277, 3844-3860]. In order to elucidate the structure-function-stability relationship of this enzyme, two residues in its active site were replaced with the residues that occur in the human l-arginine:glycine amidinotransferase (h-AGAT) at the corresponding positions (F245N and S247M), and a double variant carrying both substitutions was also created. In h-AGAT, both of these residues are critical for the function of this enzyme with regard to substrate binding, ligand-induced structural changes, and stability of the active site. In this study, we demonstrated that both single residue replacements resulted in a dramatic broadening of substrate specificity, but did not affect the kinetic mechanism. Experiments with substrate analogues indicate that donor substrates require a carboxylate group for binding. Evidence from initial velocity studies suggests that CyrA undergoes ligand-induced structural changes that involve Phe245. Stability parameters (T(opt) and T(max) ) of the CyrA variants differed from those of wild-type CyrA. Structural flexibilities of the wild type and all three variants were comparable on the basis of dynamic fluorescence quenching, indicating that changes in T(opt) are most likely attributable to localized effects within the active site. Overall, the results indicated that these two residues are essential for both stringent substrate specificity and the active site stability and flexibility of this unique cyanobacterial enzyme.     

A Triple Mutant in the Ω-loop of TEM-1 β-Lactamase Changes the Substrate Profile via a Large Conformational Change and an Altered General Base for Catalysis

β-Lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. TEM-1 is a prevalent plasmid-encoded β-lactamase in Gram-negative bacteria that efficiently catalyzes the hydrolysis of penicillins and early cephalosporins but not oxyimino-cephalosporins. A previous random mutagenesis study identified a W165Y/E166Y/P167G triple mutant that displays greatly altered substrate specificity with increased activity for the oxyimino-cephalosporin, ceftazidime, and decreased activity toward all other β-lactams tested. Surprisingly, this mutant lacks the conserved Glu-166 residue critical for enzyme function. Ceftazidime contains a large, bulky side chain that does not fit optimally in the wild-type TEM-1 active site. Therefore, it was hypothesized that the substitutions in the mutant expand the binding site in the enzyme. To investigate structural changes and address whether there is an enlargement in the active site, the crystal structure of the triple mutant was solved to 1.44 Å. The structure reveals a large conformational change of the active site Ω-loop structure to create additional space for the ceftazidime side chain. The position of the hydroxyl group of Tyr-166 and an observed shift in the pH profile of the triple mutant suggests that Tyr-166 participates in the hydrolytic mechanism of the enzyme. These findings indicate that the highly conserved Glu-166 residue can be substituted in the mechanism of serine β-lactamases. The results reveal that the robustness of the overall β-lactamase fold coupled with the plasticity of an active site loop facilitates the evolution of enzyme specificity and mechanism.     

Conformational basis for substrate recognition and regulation of catalytic activity in Staphylococcus aureus nucleoside di-phosphate kinase

Nucleoside diphosphate kinases (NDK) are characterized by high catalytic turnover rates and diverse substrate specificity. These features make this enzyme an effective activator of a pro-drug-an application that has been actively pursued for a variety of therapeutic strategies. The catalytic mechanism of this enzyme is governed by a conserved histidine that coordinates a magnesium ion at the active site. Despite substantial structural and biochemical information on NDK, the mechanistic feature of the phospho-transfer that leads to auto-phosphorylation remains unclear. While the role of the histidine residue is well documented, the other active site residues, in particular the conserved serine remains poorly characterized. Studies on some homologues suggest no role for the serine residue at the active site, while others suggest a crucial role for this serine in the regulation and quaternary association of this enzyme in some species. Here we report the biochemical features of the Staphylococcus aureus NDK and the mutant enzymes. We also describe the crystal structures of the apo-NDK, as a transition state mimic with vanadate and in complex with different nucleotide substrates. These structures formed the basis for molecular dynamics simulations to understand the broad substrate specificity of this enzyme and the role of active site residues in the phospho-transfer mechanism and oligomerization. Put together, these data suggest that concerted changes in the conformation of specific residues facilitate the stabilization of nucleotide complexes thereby enabling the steps involved in the ping-pong reaction mechanism without large changes to the overall structure of this enzyme.     

Biochemical characterization of the chondroitinase B active site

Chondroitinase B from Flavobacterium heparinum is the only known lyase that cleaves the glycosaminoglycan, dermatan sulfate (DS), as its sole substrate. A recent co-crystal structure of chondroitinase B with a disaccharide product of DS depolymerization has provided some insight into the location of the active site and suggested potential roles of some active site residues in substrate binding and catalysis. However, this co-crystal structure was not representative of the actual enzyme-substrate complex, because the disaccharide product did not have the right length or the chemical structure of the minimal substrate (tetrasaccharide) involved in catalysis. Therefore, only a limited picture of the functional role of active site residues in DS depolymerization was presented in previous structural studies. In this study, by docking a DS tetrasaccharide into the proposed active site of the enzyme, we have identified novel roles of specific active site amino acids in the catalytic function of chondroitinase B. Our conformational analysis also revealed a unique, symmetrical arrangement of active site amino acids that may impinge on the catalytic mechanism of action of chondroitinase B. The catalytic residues Lys-250, Arg-271, His-272, and Glu-333 along with the substrate binding residues Arg-363 and Arg-364 were mutated using site-directed mutagenesis, and the kinetics and product profile of each mutant were compared with recombinant chondroitinase B. Mutating Lys-250 to alanine resulted in inactivation of the enzyme, potentially attributable to the role of the residue in stabilizing the carbanion intermediate formed during enzymatic catalysis. The His-272 and Glu-333 mutants showed diminished enzymatic activity that could be indicative of a possible role for one or both residues in the abstraction of the C-5 proton from the galactosamine. In addition, the Arg-364 mutant had an altered product profile after exhaustive digestion of DS, suggesting a role for this residue in defining the substrate specificity of chondroitinase B.     

Crystal structure of human bisphosphoglycerate mutase

Bisphosphoglycerate mutase is a trifunctional enzyme of which the main function is to synthesize 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. The gene coding for bisphosphoglycerate mutase from the human cDNA library was cloned and expressed in Escherichia coli. The protein crystals were obtained and diffract to 2.5 A and produced the first crystal structure of bisphosphoglycerate mutase. The model was refined to a crystallographic R-factor of 0.200 and R(free) of 0.266 with excellent stereochemistry. The enzyme remains a dimer in the crystal. The overall structure of the enzyme resembles that of the cofactor-dependent phosphoglycerate mutase except the regions of 13-21, 98-117, 127-151, and the C-terminal tail. The conformational changes in the backbone and the side chains of some residues reveal the structural basis for the different activities between phosphoglycerate mutase and bisphosphoglycerate mutase. The bisphosphoglycerate mutase-specific residue Gly-14 may cause the most important conformational changes, which makes the side chain of Glu-13 orient toward the active site. The positions of Glu-13 and Phe-22 prevent 2,3-bisphosphoglycerate from binding in the way proposed previously. In addition, the side chain of Glu-13 would affect the Glu-89 protonation ability responsible for the low mutase activity. Other structural variations, which could be connected with functional differences, are also discussed.     

Role of the carboxyl terminal region of H(+)-ATPase (F0F1) a subunit from Escherichia coli.

The effects of amino acid substitutions in the carboxyl terminal region of the H(+)-ATPase a subunit (271 amino acid residues) of Escherichia coli were studied using a defined expression system for uncB genes coded by recombinant plasmids. The a subunits with the mutations, Tyr-263----end, Trp-231----end, Glu-219----Gln, and Arg-210----Lys (or Gln) were fully defective in ATP-dependent proton translocation, and those with Gln-252----Glu (or Leu), His-245----Glu, Pro-230----Leu, and Glu-219----His were partially defective. On the other hand, the phenotypes of the Glu-269----end, Ser-265----Ala (or end), and Tyr-263----Phe mutants were essentially similar to that of the wild-type. These results suggested that seven amino acid residues between Ser-265 and the carboxyl terminus were not required for the functional proton pathway but that all the other residues except Arg-210, Glu-219, and His-245 were required for maintaining the correct conformation of the proton pathway. The results were consistent with a report that Arg-210 is directly involved in proton translocation.     

Crystal structure of Leishmania major oligopeptidase B gives insight into the enzymatic properties of a trypanosomatid virulence factor

Oligopeptidase B (OPB) is a serine peptidase with dibasic substrate specificity. It is found in bacteria, plants, and trypanosomatid pathogens, where it has been identified as a virulence factor and potential drug target. In this study we expressed active recombinant Leishmania major OPB and provide the first structure of an oligopeptidase B at high resolution. The crystallographic study reveals that OPB comprises two domains, a catalytic and a propeller domain, linked together by a hinge region. The structure has been determined in complex with the oligopeptide, protease-inhibitor antipain, giving detailed information on the enzyme active site and extended substrate binding pockets. It shows that Glu-621 plays a critical role in the S1 binding pocket and, along with Phe-603, is largely responsible for the enzyme substrate specificity in P1. In the S2 binding pocket, Tyr-499 was shown to be important for substrate stability. The structure also allowed an investigation into the function of residues highlighted in other studies including Glu-623, which was predicted to be involved in the S1 binding pocket but is found forming an inter-domain hydrogen bond. Additional important salt bridges/hydrogen bonds between the two domains were observed, highlighting the significance of the domain interface in OPB. This work provides a foundation for the study of the role of OPBs as virulence factors in trypanosomatids. It could facilitate the development of specific OPB inhibitors with therapeutic potential by exploiting its unique substrate recognition properties as well as providing a model for OPBs in general.     

A loop involving catalytic chain residues 230-245 is essential for the stabilization of both allosteric forms of Escherichia coli aspartate transcarbamylase

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 beta-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, [S]0.5. The Arg-229----Ala enzyme still exhibits substantial homotropic cooperativity, but all cooperativity is lost in the Glu-233----Ser enzyme. The Glu-233----Ser enzyme also shows a 4-fold decrease in the carbamyl phosphate [S]0.5, while the Arg-229----Ala enzyme shows no change in the carbamyl phosphate [S]0.5 compared to the wild-type enzyme. The Glu-272 to Ser mutation results in a slight reduction in maximal activity, an increase in [S]0.5 for both aspartate and carbamyl phosphate, and reduced cooperativity. Analysis of the isolated catalytic subunits from these three mutant enzymes reveals that in each case the changes in the kinetic properties of the isolated catalytic subunit are similar to the changes caused by the mutation in the holoenzyme. PALA was able to activate the Glu-233----Ser enzyme, at low aspartate concentrations, even though the mutant holoenzyme did not exhibit any cooperativity, indicating that cooperative interactions still exist between the active sites in this enzyme. It is proposed that Glu-233 of the 240s loop helps create the high-activity-high-affinity R state by positioning the side chain of Arg-229 for aspartate binding while Glu-272 helps stabilize the low-activity-low-affinity T state by positioning the side chain of Arg-229 so that it cannot interact with aspartate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Involvement of the carboxy-terminal residue in the active site of the histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

Histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has an active site that involves His-15, which is phosphorylated to form a N delta 1-P-histidine, Arg-17, and the carboxy-terminal residue Glu-85. Mutant HPrs with alterations to the three C-terminal residues, Glu-85, Leu-84, and Glu-83, were produced by site-directed mutagenesis. The properties of these mutants were assessed by kinetic analysis of enzyme I, enzyme IImannose, enzyme IIN-acetylglucosamine, and enzyme IImannitol, and the phosphohydrolysis properties of the HPr mutants. The results show that it is the C-terminal alpha-carboxyl of Glu-85 that is involved in the active site, and this involvement may be restricted to the phosphoryl donor action of HPr. The contribution of this alpha-carboxyl group is modest as the deletion of Glu-85 resulted in the reduction of the enzyme II activity (kcat/Km) to about 33%. Removal of both Glu-85 and Leu-84 yields an HPr that is an impaired substrate of both the enzyme I and enzyme II reactions. Glu-83 appears to have no role in the active site.     

Mutation of a unique aspartate residue abolishes the catalytic activity but not substrate binding of the mouse N-methylpurine-DNA glycosylase (MPG)

N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a variety of alkylated purine adducts. Although Asp was identified as the active site residue in various DNA glycosylases based on the crystal structure, Glu-125 in human MPG (Glu-145 in mouse MPG) was recently proposed to be the catalytic residue. Mutational analysis for all Asp residues in a truncated, fully active MPG protein showed that only Asp-152 (Asp-132 in the human protein), which is located near the active site, is essential for catalytic activity. However, the substrate binding was not affected in the inactive Glu-152, Asn-152, and Ala-152 mutants. Furthermore, mutation of Asp-152 did not significantly affect the intrinsic tryptophan fluorescence of the enzyme and the far UV CD spectra, although a small change in the near UV CD spectra of the mutants suggests localized conformational change in the aromatic residues. We propose that in addition to Glu-145 in mouse MPG, which functions as the activator of a water molecule for nucleophilic attack, Asp-152 plays an essential role either by donating a proton to the substrate base and, thus, facilitating its release or by stabilizing the steric configuration of the active site pocket.     

Identification of amino acid residues essential for the substrate specificity of Flavobacterium sp. endo-beta-N-acetylglucosaminidase

The gene encoding the endo-beta-N-acetylglucosaminidase from Flavobacterium sp. (Endo-Fsp) was sequenced. The Endo-Fsp gene was overexpressed in Escherichia coli cells, and was purified from inclusion bodies after denaturation by 8 M urea. The renatured Endo-Fsp had the same optimum pH and substrate specificity as the native enzyme. Endo-Fsp had 60% sequence identity with the endo-beta-N-acetylglucosaminidase from Streptomyces plicatus (Endo-H), and the putative catalytic residues were conserved. Site-directed mutagenesis was done at conserved residues based on the three-dimensional structure and mutagenesis of Endo-H. The mutant of Glu-128, corresponding to Glu-132 in Endo-H and identified as an active site residue, was inactivated. Mutagenesis around the predicted active site of Endo-Fsp reduced the enzymatic activity. Moreover, the hydrolytic activity toward hybrid-type oligosaccharides was decreased compared to that toward high-mannose type oligosaccharides by mutagenesis of Asp-126 and Asp-127. Therefore, site-directed mutagenesis of some of these conserved residues indicates that the predicted active sites are essential to the enzymatic activity of Endo-Fsp, and may have similar roles in catalysis as their counterparts in Endo-H.     

Pinpointing biphenyl dioxygenase residues that are crucial for substrate interaction

Three regions of the biphenyl dioxygenase (BDO) of Burkholderia sp. strain LB400 have previously been shown to significantly influence the interaction between enzyme and substrates at the active site. For a further discrimination within these regions, we investigated the effects of 23 individual amino acid exchanges. The regiospecificity of substrate dioxygenation was used as a sensitive means to monitor changes in the steric-electronic structure of the active site. Replacements of residues that, according to a model of the BDO three-dimensional structure, directly interact with substrates in most, but not all, cases (Met231, Phe378, and Phe384) very strongly altered this parameter (by factors of >7). On the other hand, a number of amino acids (Ile243, Ile326, Phe332, Pro334, and Trp392) which have no contacts with substrates also strongly changed the site preference of dioxygenation (by factors of between 2.6 and 3.5). This demonstrates that residues which had not been predicted to be influential can play a pivotal role in BDO specificity.     

Starch- and glycogen-debranching and branching enzymes: Prediction of structural features of the catalytic (β/α)8-barrel domain and evolutionary relationship to other amylolytic enzymes

Sequence alignment and structure prediction are used to locate catalytic α-amylase-type (β/α)₈-barrel domains and the positions of their β-strands and α-helices in isoamylase, pullulanase, neopullulanase, α-amylase-pullulanase, dextran glucosidase, branching enzyme, and glycogen branching enzymes—all enzymes involved in hydrolysis or synthesis of α-1,6-glucosidic linkages in starch and related polysaccharides. This has allowed identification of the transferase active site of the glycogen debranching enzyme and the locations of β ? α loops making up the active sites of all enzymes studied. Activity and specificity of the enzymes are discussed in terms of conserved amino acid residues and loop variations. An evolutionary distance tree of 47 amylolytic and related enzymes is built on 37 residues representing the four best conserved β-strands of the barrel. It exhibits clusters of enzymes close in specificity, with the branching and glycogen debranching enzymes being the most distantly related.     

The molecular architecture of human N-acetylgalactosamine kinase

Galactokinase plays a key role in normal galactose metabolism by catalyzing the conversion of alpha-d-galactose to galactose 1-phosphate. Within recent years, the three-dimensional structures of human galactokinase and two bacterial forms of the enzyme have been determined. Originally, the gene encoding galactokinase in humans was mapped to chromosome 17. An additional gene, encoding a protein with sequence similarity to galactokinase, was subsequently mapped to chromosome 15. Recent reports have shown that this second gene (GALK2) encodes an enzyme with greater activity against GalNAc than galactose. This enzyme, GalNAc kinase, has been implicated in a salvage pathway for the reutilization of free GalNAc derived from the degradation of complex carbohydrates. Here we report the first structural analysis of a GalNAc kinase. The structure of the human enzyme was solved in the presence of MnAMPPNP and GalNAc or MgATP and GalNAc (which resulted in bound products in the active site). The enzyme displays a distinctly bilobal appearance with its active site wedged between the two domains. The N-terminal region is dominated by a seven-stranded mixed beta-sheet, whereas the C-terminal motif contains two layers of anti-parallel beta-sheet. The overall topology displayed by GalNAc kinase places it into the GHMP superfamily of enzymes, which generally function as small molecule kinases. From this investigation, the geometry of the GalNAc kinase active site before and after catalysis has been revealed, and the determinants of substrate specificity have been defined on a molecular level.     

Crystal structure of the bovine α-chymotrypsin:kunitz inhibitor complex. An example of multiple protein:protein recognition sites

The crystal structure of bovine α-chymotrypsin (α-CHT) in complex with the bovine basic pancreatic trypsin inhibitor (BPTI) has been solved and refined at 2.8 Å resolution (R-factor=0.18). The proteinase:inhibitor complex forms a compact dimer (two α-CHT and two BPTI molecules), which may be stabilized by surface-bound sulphate ions, in the crystalline state. Each BPTI molecule, at opposite ends, is contacting both proteinase molecules in the dimer, through the reactive site loop and through residues next to the inhibitor's C-terminal region. Specific recognition between α-CHT and BPTI occurs at the (re)active site interface according to structural rules inferred from the analysis of homologous serine proteinase:inhibitor complexes. Lys15, the P₁ residue of BPTI, however, does not occupy the α-CHT S₁ specificity pocket, being hydrogen bonded to backbone atoms of the enzyme surface residues Gly216 and Ser217. © 1997 John Wiley & Sons, Ltd.     

Studies of specificity and catalysis in trypsin by structural analysis of site-directed mutants

We are probing the determinants of catalytic function and substrate specificity in serine proteases by kinetic and crystallographic characterization of genetically engineered site-directed mutants of rat trypsin. The role of the aspartyl residue at position 102, common to all members of the serine protease family, has been tested by substitution with asparagine. In the native enzyme, Asp102 accepts a hydrogen bond from the catalytic base His57, which facilitates the transfer of a proton from the enzyme nucleophile Ser195 to the substrate leaving group. At neutral pH, the mutant is four orders of magnitude less active than the naturally occurring enzyme, but its binding affinity for model substrates is virtually undiminished. Crystallographic analysis reveals that Asn102 donates a hydrogen bond to His57, forcing it to act as donor to Ser195. Below pH 6, His57 becomes statistically disordered. Presumably, the di-protonated population of histidyl side chains are unable to hydrogen bond to Asn102. Steric conflict may cause His57 to rotate away from the catalytic site. These results suggest that Asp102 not only provides inductive and orientation effects, but also stabilizes the productive tautomer of His57. Three experiments were carried out to alter the substrate specificity of trypsin. Glycine residues at positions 216 and 226 in the substrate-binding cavity were replaced by alanine residues in order to differentially affect lysine and arginine substrate binding. While the rate of catalysis by the mutant enzymes was reduced in the mutant enzymes, their substrate specificity was enhanced relative to trypsin. The increased specificity was caused by differential effects on the catalytic activity towards arginine and lysine substrates. The Gly----Ala substitution at 226 resulted in an altered conformation of the enzyme which is converted to an active trypsin-like conformation upon binding of a substrate analog. In a third experiment, Lys189, at the bottom of the specificity pocket, was replaced with an aspartate with the expectation that specificity of the enzyme might shift to aspartate. The mutant enzyme is not capable of cleaving at Arg and Lys or Asp, but shows an enhanced chymotrypsin-like specificity. Structural investigations of these mutants are in progress.     

Modification of near active site residues in organophosphorus hydrolase reduces metal stoichiometry and alters substrate specificity

Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a dimeric, bacterial enzyme that detoxifies many organophosphorus neurotoxins by hydrolyzing a variety of phosphonate bonds. The histidinyl residues at amino acid positions 254 and 257 are located near the bimetallic active site present in each monomer. It has been proposed that these residues influence catalysis by interacting with active site residues and the substrate in the binding pocket. We replaced the histidine at position 254 with arginine (H254R) and the one at position 257 with leucine (H257L) independently to form the single-site-modified enzymes. The double modification was also constructed to incorporate both changes (H254R/H257L). Although native OPH has two metals at each active site (four per dimer), all three of these altered enzymes possessed only two metals per dimer while retaining considerable enzymatic activity for the preferred phosphotriester (P-O bond) substrate, paraoxon (5-100% kcat). The three altered enzymes achieved a 2-30-fold increase in substrate specificity (kcat/Km) for demeton S (P-S bond), an analogue for the chemical warfare agent VX. In contrast, the substrate specificity for diisopropyl fluorophosphonate (P-F bond) was substantially decreased for each of these enzymes. In addition, H257L and H254R/H257L showed an 11- and 18-fold increase, respectively, in specificity for NPPMP, the analogue for the chemical warfare agent soman. These results demonstrate the ability to significantly enhance the specificity of OPH for various substrates by site-specific modifications, and it is suggested that changes in metal requirements may affect these improved catalytic characteristics by enhancing structural flexibility and improving access of larger substrates to the active site, while simultaneously decreasing the catalytic efficiency for smaller substrates.     

Investigating the roles of putative active site residues in the oxalate decarboxylase from Bacillus subtilis

Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO(2) and formate using a catalytic mechanism that remains poorly understood. The Bacillus subtilis enzyme is composed of two cupin domains, each of which contains Mn(II) coordinated by four conserved residues. We have measured heavy atom isotope effects for a series of Bacillus subtilis OxDC mutants in which Arg-92, Arg-270, Glu-162, and Glu-333 are conservatively substituted in an effort to define the functional roles of these residues. This strategy has the advantage that observed isotope effects report directly on OxDC molecules in which the active site manganese center(s) is (are) catalytically active. Our results support the proposal that the N-terminal Mn-binding site can mediate catalysis, and confirm the importance of Arg-92 in catalytic activity. On the other hand, substitution of Arg-270 and Glu-333 affects both Mn(II) incorporation and the ability of Mn to bind to the OxDC mutants, thereby precluding any definitive assessment of whether the metal center in the C-terminal domain can also mediate catalysis. New evidence for the importance of Glu-162 in controlling metal reactivity has been provided by the unexpected observation that the E162Q OxDC mutant exhibits a significantly increased oxalate oxidase and a concomitant reduction in decarboxylase activities relative to wild type OxDC. Hence the reaction specificity of a catalytically active Mn center in OxDC can be perturbed by relatively small changes in local protein environment, in agreement with a proposal based on prior computational studies.     

Site-directed alteration of the active-site residues of histidine decarboxylase from Clostridium perfringens

To clarify the mechanism of biogenesis and catalysis by the pyruvoyl-dependent histidine decarboxylase (HisDCase) from Clostridium perfringens, 12 mutant genes encoding amino acid substitutions at the active site of this enzyme were constructed and expressed in Escherichia coli. The resulting mutant proteins were purified to homogeneity, characterized, and subjected to kinetic analysis. The results (a) exclude all polar amino acid residues in the active site except Glu-214 as donor of the proton that replaces the carboxyl group of histidine during decarboxylation and, since E214I and E214H are nearly inactive, indicate that Glu-214 is the essential proton donor; (b) demonstrate the importance to substrate binding of hydrophobic interactions between Phe-98, Ile-74, and the imidazole ring of histidine, and of hydrogen bonding between Asp-78 and N2 of the substrate; and (c) demonstrate a significant unidentified role for Glu-81 in the maintenance of the active-site structure. The proposed roles of these amino acid residues are consistent with those assigned on the basis of crystallographic evidence to the corresponding residues at the active site of the related HisDCase from Lactobacillus 30a [Gallagher, T., Snell, E. E., & Hackert, M. L. (1989) J. Biol. Chem. 264, 12737-12743]. Of the residues altered, only Ser-97 was essential for the autocatalytic serinolysis reaction by which this HisDCase, (alpha beta)6, is derived from its inactive, pyruvate-free precursor, proHisDCase, pi 6.(ABSTRACT TRUNCATED AT 250 WORDS)     

His230 of serine hydroxymethyltransferase facilitates the proton abstraction step in catalysis.

The three-dimensional structures of rabbit and human liver cytosolic serine hydroxymethyltransferase revealed that H231 interacts with the O3' of pyridoxal-5'-phosphate and other residues at the active site such as S203, K257, H357 and R402 (numbering as per the human enzyme). This and the conserved nature of H231 in all serine hydroxymethyltransferases highlights its importance in catalysis and/or maintenance of oligomeric structure of the enzyme. In an attempt to decipher the role of H230 (H231 of the human enzyme) in the catalytic mechanism and/or maintenance of oligomeric structure of sheep liver serine hydroxymethyltransferase, the residue was mutated to arginine, phenylalanine, alanine, asparagine or tyrosine. Our results suggest that the nature of the amino acid substitution has a marked effect on the catalytic activity of the enzyme. H230R and H230F mutant proteins were completely inactive, dimeric and did not bind pyridoxal-5'-phosphate. On the other hand, mutation to alanine and asparagine retained the oligomeric structure and ability to bind pyridoxal-5'-phosphate. These mutants had only 2-3% catalytic activity. The side reactions like transamination and 5,6,7, 8-tetrahydrofolate independent aldol cleavage were much more severely affected. They were able to form the external aldimine with glycine and serine but the quinonoid intermediate was not observed upon the addition of 5,6,7,8-tetrahydrofolate. Mutation to tyrosine did not affect the oligomeric structure and pyridoxal-5'-phosphate binding. The H230Y enzyme was 10% active and showed a correspondingly lower amount of quinonoid intermediate. The kcat / Km values for L-serine and Lallothreonine were 10-fold and 174-fold less for this mutant enzyme compared to the wild-type protein. These results suggest that H230 is involved in the step prior to the formation of the quinonoid intermediate, possibly in orienting the pyridine ring of the cofactor, in order to facilitate effective proton abstraction.