High catalytic activity of alanine racemase from psychrophilic Bacillus psychrosaccharolyticus at high temperatures in the presence of pyridoxal 5'-phosphate

We examined the effect of the pyridoxal 5'-phosphate (PLP) cofactor on the activity and stability of the psychrophilic alanine racemase, having a high catalytic activity at low temperature, from Bacillus psychrosaccharolyticus at high temperatures. The decrease in the enzyme activity at incubation temperatures over 40 degrees C was consistent with the decrease in the amount of bound PLP. Unfolding of the enzyme at temperatures above 40 degrees C was suppressed in the presence of PLP. In the presence of 0.125 mM PLP, the specific activity of the psychrophilic enzyme was higher than that of a thermophilic alanine racemase, having a high catalytic activity at high temperature, from Bacillus stearothermophilus even at 60 degrees C.     

Structure and function of psychrophilic alanine racemase

We describe the structure and function of psychrophilic alanine racemases from Bacillus psychrosaccharolyticus and Pseudomonas fluorescens. These enzymes showed high catalytic activities even at 0°C and were extremely labile at temperatures over 35°C. The enzymes were also found to be less resistant to organic solvents than alanine racemases from thermophilic and mesophilic bacteria, both in vivo and in vitro. Both enzymes have a dimeric structure and contain 2 mol of pyridoxal 5′-phosphate (PLP) per mol as a coenzyme. The enzyme from B. psychrosaccharolyticus was found to have a markedly large K_m value (5.0 μM) for PLP in comparison with other reported alanine racemases, and was stable at temperatures up to 50°C in the presence of excess amounts of PLP. The dissociation of PLP from the P. fluorescens enzyme may trigger the unfolding of the secondary structure. The enzyme from B. psychrosaccharolyticus has a distinguishing hydrophilic region around residue no. 150 in its deduced amino acid sequence, whereas the corresponding regions of other Bacillus alanine racemases are hydrophobic. The position of this region in the three dimensional structure of this enzyme was predicted to be in a surface loop surrounding the active site. This hydrophilic region may interact with solvent, reduce the compactness of the active site, and destabilize the enzyme.     

Stereochemistry of the hydrogen abstraction from pyridoxamine phosphate catalyzed by alanine racemase of Bacillus stearothermophilus

Alanine racemase of Bacillus stearothermophilus catalyzes transamination as a side reaction. Stereospecificity for the hydrogen abstraction from C-4′ of pyridoxamine 5′-phosphate occurring in the latter half transamination was examined. Both apo-wild-type and apo-fragmentary alanine racemases abstracted approximately 20 and 80% of tritium from the stereospecifically-labeled (4′S)- and (4′R)-[4′-³H]PMP, respectively, in the presence of pyruvate. Alanine racemase catalyzes the abstraction of both 4′S- and 4′R-hydrogen like amino acid racemase with broad substrate specificity. However, R-isomer preference is a characteristic property of alanine racemase.     

Characterization of psychrophilic alanine racemase from Bacillus psychrosaccharolyticus

A psychrophilic alanine racemase gene from Bacillus psychrosaccharolyticus was cloned and expressed in Escherichia coli SOLR with a plasmid pYOK3. The gene starting with the unusual initiation codon GTG showed higher preference for codons ending in A or T. The enzyme purified to homogeneity showed the high catalytic activity even at 0 degrees C and was extremely labile over 35 degrees C. The enzyme was found to have a markedly large Km value (5.0 microM) for the pyridoxal 5'-phosphate (PLP) cofactor in comparison with other reported alanine racemases, and was stabilized up to 50 degrees C in the presence of excess amounts of PLP. The low affinity of the enzyme for PLP may be related to the thermolability, and may be related to the high catalytic activity, initiated by the transaldimination reaction, at low temperature. The enzyme has a distinguishing hydrophilic region around the residue no. 150 in the deduced amino acid sequence (383 residues), whereas the corresponding regions of other Bacillus alanine racemases are hydrophobic. The position of the region in the three dimensional structure of C atoms of the enzyme was predicted to be in a surface loop surrounding the active site. The region may interact with solvent and reduce the compactness of the active site.     

Cold-active citrate synthase: mutagenesis of active-site residues.

A comparison of the crystal structure of the dimeric enzyme citrate synthase from the psychrophilic Arthrobacter strain DS2-3R with that of the structurally homologous enzyme from the hyperthermophilic Pyrococcus furiosus reveals a significant difference in the accessibility of their active sites to substrates. In this work, we investigated the possible role in cold activity of the greater accessibility of the Arthrobacter citrate synthase. By site-directed mutagenesis, we replaced two alanine residues at the entrance to the active site with an arginine and glutamate residue, respectively, as found in the equivalent positions of the Pyrococcus enzyme Also, we introduced a loop into the active site of the psychrophilic citrate synthase, again mimicking the situation in the hyperthermophilic enzyme. Analysis of the thermoactivity and thermostability of the mutant enzymes reveals that cold activity is not significantly compromised by the mutations, but rather the affinity for one of the substrates, acetyl-CoA, is dramatically increased. Moreover, one mutant (Loop insertion/K313L/A361R) has an increased thermostability but a reduced temperature optimum for catalytic activity. This unexpected relationship between stability and activity is discussed with respect to the nature of the dependence of catalytic activity on temperature.     

Purification and characterization of alanine racemase from hepatopancreas of black-tiger prawn, Penaeus monodon

Alanine racemase has been purified to homogeneity from the hepatopancreas of the black tiger prawn, Panaeus mondon. The enzyme depends on pyridoxal 5′-phosphate and consists of two subunits with an identical molecular weight of 41,000. V_max and K_m values for -alanine are 460 μmol/min/mg and 50 mM, and those for -alanine are 94 μmol/min/mg and 24 mM, respectively. The enzyme is highly specific toward alanine. Among other amino acids examined, only serine served as a substrate: -serine was racemized at a rate of approximately 0.5% of that of -alanine. The prawn enzyme is immunochemically distinguishable from the enzymes of Bacillus stearothermophilus and Schizosaccharomyces pombe, which resemble each other. The prawn enzyme is activated and stabilized by the presence of monovalent anions including chloride. This is consistent with the previous hypothesis (e.g. E. Fujita, E. Okuma, H. Abe, Comp. Biochem. Physiol. 116A (1997) 83–87) that -alanine serves as an osmoregulator in marine and euryhaline animals.     

Effect of a Y265F mutant on the transamination-based cycloserine inactivation of alanine racemase

The requirement for d-alanine in the peptidoglycan layer of bacterial cell walls is fulfilled in part by alanine racemase (EC 5.1.1.1), a pyridoxal 5'-phosphate (PLP)-assisted enzyme. The enzyme utilizes two antiparallel bases focused at the C(alpha) position and oriented perpendicular to the PLP ring to facilitate the equilibration of alanine enantiomers. Understanding how this two-base system is utilized and controlled to yield reaction specificity is therefore a potential means for designing antibiotics. Cycloserine is a known alanine racemase suicide substrate, although its mechanism of inactivation is based on transaminase chemistry. Here we characterize the effects of a Y265F mutant (Tyr265 acts as the catalytic base in the l-isomer case) of Bacillus stearothermophilus alanine racemase on cycloserine inactivation. The Y265F mutant reduces racemization activity 1600-fold [Watanabe, A., Yoshimura, T., Mikami, B., and Esaki, N. (1999) J. Biochem. 126, 781-786] and only leads to formation of the isoxazole end product (the result of the transaminase pathway) in the case of d-cycloserine, in contrast to results obtained using the wild-type enzyme. l-Cycloserine, on the other hand, utilizes a number of alternative pathways in the absence of Y265, emphasizing the importance of Y265 in both the inactivation and racemization pathway. In combination with the kinetics of inactivation, these results suggest roles for each of the two catalytic bases in racemization and inactivation, as well as the importance of Y265 in "steering" the chemistry to favor one pathway over another.     

Catalytic mechanism of serine racemase from Dictyostelium discoideum

The eukaryotic serine racemase from Dictyostelium discoideum is a fold-type II pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes racemization and dehydration of both isomers of serine. In the present study, the catalytic mechanism and role of the active site residues of the enzyme were examined by site-directed mutagenesis. Mutation of the PLP-binding lysine (K56) to alanine abolished both serine racemase and dehydrase activities. Incubation of d- and l-serine with the resultant mutant enzyme, K56A, resulted in the accumulation of PLP-serine external aldimine, while less amounts of pyruvate, α-aminoacrylate, antipodal serine and quinonoid intermediate were formed. An alanine mutation of Ser81 (S81) located on the opposite side of K56 against the PLP plane converted the enzyme from serine racemase to l-serine dehydrase; S81A showed no racemase activity and had significantly reduced d-serine dehydrase activity, but it completely retained its l-serine dehydrase activity. Water molecule(s) at the active site of the S81A mutant enzyme probably drove d-serine dehydration by abstracting the α-hydrogen in d-serine. Our data suggest that the abstraction and addition of α-hydrogen to l- and d-serine are conducted by K56 and S81 at the si- and re-sides, respectively, of PLP.     

Paradoxical thermostable enzymes from psychrophile: molecular characterization and potentiality for biotechnological application

NAD(P)⁺-dependent aldehyde dehydrogenase (EC 1.2.1.5) and aspartase (EC 4.3.1.1) in the cells of an atypical psychrophile from Antarctic seawater, Cytophaga sp. KUC-1, were paradoxically thermostable, although they derived from a psychrophile. Both enzymes showed the highest activity at about 55 °C, and also active even under cold conditions. The enzymes contained more Ile residues than the enzymes from mesophiles. The Ile/Ile + Val + Leu ratio of the Cytophaga thermostable enzymes was much higher than that of the enzymes from mesophiles. As compared with the enzymes from other microorganisms, the Cytophaga thermostable enzymes have the structural differences in the C-terminal region of the enzymes. Therefore, the C-terminal region might be important for the paradoxical thermostability of the enzymes. The psychrophilic microorganism produces not only psychrophilic enzyme, but thermostable enzyme with psychrophilicity. Therefore, the psychrophilic microorganism is one of the candidates for isolation of novel biocatalysts, which have potential for various industrial applications.     

Isolation of a Bacillus stearothermophilus mutant exhibiting increased thermostability in its restriction endonuclease.

A procedure was developed for the selection of spontaneous mutants of Bacillus stearothermophilus NUB31 that are more efficient than the wild type in the restriction of phage at elevated temperatures. Inactivation studies revealed that two mutants contained a more thermostable restriction enzyme and one mutant contained three times more enzyme than the wild type. The restriction endonucleases from the wild type and one of the mutants were purified to apparent homogeneity. The mutant enzyme was more thermostable than the wild-type enzyme. The subunit molecular weight, amino acid composition, N-terminal and C-terminal amino acid residues, tryptic peptide map, and catalytic properties of the two enzymes were determined. The two enzymes have similar catalytic properties, but the molecular size of the mutant enzyme is approximately 6 to 7 kilodaltons larger than that of the wild-type enzyme. The mutant enzyme contains 54 additional amino acid residues, of which 26 to 28 are aspartate/asparagine, 8 to 15 are glutamate/glutamine, and 8 to 9 are tyrosine residues. The two enzymes contained similar amounts of the other amino acids, identical N-terminal residues, and different C-terminal residues. Tryptic peptide analyses revealed a high degree of homology between the two enzymes. The increased thermostability observed in the mutant enzyme appears to have been achieved by a mutation that resulted in the addition of amino acid residues to the wild-type enzyme. A number of mechanisms are discussed that could account for the observed difference between the mutant and wild-type enzymes.     

Crystal Structures of Lysine-Preferred Racemases,the Non-Antibiotic Selectable Markers for Transgenic Plants

Lysine racemase, a pyridoxal 5′-phosphate (PLP)-dependent amino acid racemase that catalyzes the interconversion of lysine enantiomers, is valuable to serve as a novel non-antibiotic selectable marker in the generation of transgenic plants. Here, we have determined the first crystal structure of a lysine racemase (Lyr) from Proteus mirabilis BCRC10725, which shows the highest activity toward lysine and weaker activity towards arginine. In addition, we establish the first broad-specificity amino acid racemase (Bar) structure from Pseudomonas putida DSM84, which presents not only the highest activity toward lysine but also remarkably broad substrate specificity. A complex structure of Bar-lysine is also established here. These structures demonstrate the similar fold of alanine racemase, which is a head-to-tail homodimer with each protomer containing an N-terminal (α/β)₈ barrel and a C-terminal β-stranded domain. The active-site residues are located at the protomer interface that is a funnel-like cavity with two catalytic bases, one from each protomer, and the PLP binding site is at the bottom of this cavity. Structural comparisons, site-directed mutagenesis, kinetic, and modeling studies identify a conserved arginine and an adjacent conserved asparagine that fix the orientation of the PLP O3 atom in both structures and assist in the enzyme activity. Furthermore, side chains of two residues in α-helix 10 have been discovered to point toward the cavity and define the substrate specificity. Our results provide a structural foundation for the design of racemases with pre-determined substrate specificity and for the development of the non-antibiotic selection system in transgenic plants.     

腾冲嗜热厌氧菌丙氨酸消旋酶底物通道氨基酸位点的功能

【目的】通过定点突变探究腾冲嗜热厌氧菌MB4中生物合成型丙氨酸消旋酶Tt Alr底物通道内氨基酸位点A172和S173的功能。【方法】利用定点突变PCR技术构建突变体,通过亲和层析法纯化酶蛋白,采用D-氨基酸氧化酶偶联法检测各突变蛋白的活性及其稳定性。【结果】通过定点突变PCR成功得到8个突变体,酶学特性分析发现,A172位点突变为丝氨酸(S)后酶蛋白的相对活性有所提升,但含有该位点突变的酶蛋白稳定性均大幅下降;S173位点突变为天门冬氨酸(D)后导致突变体蛋白的最适反应温度提升了15°C,半衰期大幅延长,但相对活性明显下降。【结论】丙氨酸消旋酶Tt Alr底物通道内A172和S173位点均是影响酶蛋白催化活性和稳定性的关键位点。     

Reaction mechanism of alanine racemase from Bacillus stearothermophilus: x-ray crystallographic studies of the enzyme bound with N-(5'-phosphopyridoxyl)alanine

The crystal structures of alanine racemase bound with reaction intermediate analogs, N-(5'-phosphopyridoxyl)-L-alanine (PLP-L-Ala) and N-(5'-phosphopyridoxyl)-D-alanine (PLP-D-Ala), were determined at 2.0-A resolution with the crystallographic R factor of 17.2 for PLP-L-Ala and 16.9 for PLP-D-Ala complexes. They were quite similar not only to each other but also to the structure of the native pyridoxal 5'-phosphate (PLP)-form enzyme; root mean square deviations at Calpha among the three structures were less than 0.28 A. The side chains of the amino acid residues around the PLP-L-Ala and PLP-D-Ala were virtually superimposable on each other as well as on those around PLP of the native holoenzyme. The alpha-hydrogen of the alanine moiety of PLP-L-Ala was located near the OH of Tyr(265)', whereas that of PLP-D-Ala was near the NZ of Lys(39). These support the previous findings that Tyr(265)' and Lys(39) are the catalytic bases removing alpha-hydrogen from L- and D-alanine, respectively. The prerequisite for this two-base mechanism is that the alpha-proton abstracted from the substrate is transferred (directly or indirectly) between the NZ of Lys(39) and the OH of Tyr(265'); otherwise the enzyme reaction stops after a single turnover. Only the carboxylate oxygen atom of either PLP-Ala enantiomer occurred at a reasonable position that can mediate the proton transfer; neither the amino acid side chains nor the water molecules were located in the vicinity. Therefore, we propose a mechanism of alanine racemase reaction in which the substrate carboxyl group directly participates in the catalysis by mediating the proton transfer between the two catalytic bases, Lys(39) and Tyr(265)'. The results of molecular orbital calculation also support this mechanism.     

The linker region plays a key role in the adaptation to cold of the cellulase from an Antarctic bacterium

The psychrophilic cellulase, Cel5G, from the Antarctic bacterium Pseudoalteromonas haloplanktis is composed of a catalytic module (CM) joined to a carbohydrate-binding module (CBM) by an unusually long, extended and flexible linker region (LR) containing three loops closed by three disulfide bridges. To evaluate the possible role of this region in cold adaptation, the LR was sequentially shortened by protein engineering, successively deleting one and two loops of this module, whereas the last disulfide bridge was also suppressed by replacing the last two cysteine residue by two alanine residues. The kinetic and thermodynamic properties of the mutants were compared with those of the full-length enzyme, and also with those of the cold-adapted CM alone and with those of the homologous mesophilic enzyme, Cel5A, from Erwinia chrysanthemi. The thermostability of the mutated enzymes as well as their relative flexibility were evaluated by differential scanning calorimetry and fluorescence quenching respectively. The topology of the structure of the shortest mutant was determined by SAXS (small-angle X-ray scattering). The data indicate that the sequential shortening of the LR induces a regular decrease of the specific activity towards macromolecular substrates, reduces the relative flexibility and concomitantly increases the thermostability of the shortened enzymes. This demonstrates that the long LR of the full-length enzyme favours the catalytic efficiency at low and moderate temperatures by rendering the structure not only less compact, but also less stable, and plays a crucial role in the adaptation to cold of this cellulolytic enzyme.     

L-allo-threonine aldolase from Aeromonas jandaei DK-39: gene cloning, nucleotide sequencing, and identification of the pyridoxal 5'-phosphate-binding lysine residue by site-directed mutagenesis.

We have isolated the gene encoding L-allo-threonine aldolase (L-allo-TA) from Aeromonas jandaei DK-39, a pyridoxal 5'-phosphate (PLP)-dependent enzyme that stereospecifically catalyzes the interconversion of L-allo-threonine and glycine. The gene contains an open reading frame consisting of 1,014 nucleotides corresponding to 338 amino acid residues. The protein molecular weight was estimated to be 36,294, which is in good agreement with the subunit molecular weight of the enzyme determined by polyacrylamide gel electrophoresis. The enzyme was overexpressed in recombinant Escherichia coli cells and purified to homogeneity by one hydrophobic column chromatography step. The predicted amino acid sequence showed no significant similarity to those of the currently known PLP-dependent enzymes but displayed 40 and 41% identity with those of the hypothetical GLY1 protein of Saccharomyces cerevisiae and the GLY1-like protein of Caenorhabditis elegans, respectively. Accordingly, L-allo-TA might represent a new type of PLP-dependent enzyme. To determine the PLP-binding site of the enzyme, all of the three conserved lysine residues of L-allo-TA were replaced by alanine by site-directed mutagenesis. The purified mutant enzymes, K51A and K224A, showed properties similar to those of the wild type, while the mutant enzyme K199A was catalytically inactive, with corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys199 of L-allo-TA probably functions as an essential catalytic residue forming an internal Schiff base with PLP of the enzyme to catalyze the reversible aldol reaction.     

Crystal structure of aspartate racemase from Pyrococcus horikoshii OT3 and its implications for molecular mechanism of PLP-independent racemization

There exists a d-enantiomer of aspartic acid in lactic acid bacteria and several hyperthermophilic archaea, which is biosynthesized from the l-enantiomer by aspartate racemase. Aspartate racemase is a representative pyridoxal 5'-phosphate (PLP)-independent amino acid racemase. The "two-base" catalytic mechanism has been proposed for this type of racemase, in which a pair of cysteine residues are utilized as the conjugated catalytic acid and base. We have determined the three-dimensional structure of aspartate racemase from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 at 1.9 A resolution by X-ray crystallography and refined it to a crystallographic R factor of 19.4% (R(free) of 22.2%). This is the first structure reported for aspartate racemase, indeed for any amino acid racemase from archaea. The crystal structure revealed that this enzyme forms a stable dimeric structure with a strong three-layered inter-subunit interaction, and that its subunit consists of two structurally homologous alpha/beta domains, each containing a four-stranded parallel beta-sheet flanked by six alpha-helices. Two strictly conserved cysteine residues (Cys82 and Cys194), which have been shown biochemically to act as catalytic acid and base, are located on both sides of a cleft between the two domains. The spatial arrangement of these two cysteine residues supports the "two-base" mechanism but disproves the previous hypothesis that the active site of aspartate racemase is located at the dimeric interface. The structure revealed a unique pseudo mirror-symmetry in the spatial arrangement of the residues around the active site, which may explain the molecular recognition mechanism of the mirror-symmetric aspartate enantiomers by the non-mirror-symmetric aspartate racemase.     

The 1.9 A crystal structure of alanine racemase from Mycobacterium tuberculosis contains a conserved entryway into the active site

We report the crystal structure of alanine racemase from Mycobacterium tuberculosis (Alr(Mtb)) at 1.9 A resolution. In our structure, Alr(Mtb) is found to be a dimer formed by two crystallographically different monomers, each comprising 384 residues. The domain makeup of each monomer is similar to that of Bacillus and Pseudomonas alanine racemases and includes both an alpha/beta-barrel at the N-terminus and a C-terminus primarily made of beta-strands. The hinge angle between these two domains is unique for Alr(Mtb), but the active site geometry is conserved. In Alr(Mtb), the PLP cofactor is covalently bound to the protein via an internal aldimine bond with Lys42. No guest substrate is noted in its active site, although some residual electron density is observed in the enzyme's active site pocket. Analysis of the active site pocket, in the context of other known alanine racemases, allows us to propose the inclusion of conserved residues found at the entrance to the binding pocket as additional targets in ongoing structure-aided drug design efforts. Also, as observed in other alanine racemase structures, PLP adopts a conformation that significantly distorts the planarity of the extended conjugated system between the PLP ring and the internal aldimine bond.     

Mutational analysis of Thermus caldophilus GK24 beta-glycosidase: role of His119 in substrate binding and enzyme activity

Three amino acid residues (His119, Glu164, and Glu338) in the active site of Thermus caldophilus GK24 beta- glycosidase (Tca beta-glycosidase), a family 1 glycosyl hydrolase, were mutated by site-directed mutagenesis. To verify the key catalytic residues, Glu164 and Glu338 were changed to Gly and Gln, respectively. The E164G mutation resulted in drastic reductions of both beta-galactosidase and beta-glucosidase activities, and the E338Q mutation caused complete loss of activity, confirming that the two residues are essential for the reaction process of glycosidic linkage hydrolysis. To investigate the role of His119 in substrate binding and enzyme activity, the residue was substituted with Gly. The H119G mutant showed 53-fold reduced activity on 5 mM p-nitrophenyl beta-Dgalactopyranoside, when compared with the wild type; however, both the wild-type and mutant enzymes showed similar activity on 5 mM p-nitrophenyl beta-D-glucopyranoside at 75degreeC. Kinetic analysis with p-nitrophenyl beta-D-galactopyranoside revealed that the kcat value of the H119G mutant was 76.3-fold lower than that of the wild type, but the Km of the mutant was 15.3-fold higher than that of the wild type owing to the much lower affinity of the mutant. Thus, the catalytic efficiency (kcat/Km) of the mutant decreased to 0.08% to that of the wild type. The kcat value of the H119G mutant for p-nitrophenyl beta- D-glucopyranoside was 5.1-fold higher than that of the wild type, but the catalytic efficiency of the mutant was 2.5% of that of the wild type. The H119G mutation gave rise to changes in optima pH (from 5.5-6.5 to 5.5) and temperature (from 90 degrees C to 80-85 degrees C). This difference of temperature optima originated in the decrease of H119G's thermostability. These results indicate that His119 is a crucial residue in beta- galactosidase and beta-glucosidase activities and also influences the enzyme's substrate binding affinity and thermostability.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, XI. Engineering thermostability and activity of lactate dehydrogenases from bacilli

An extensive comparative structural analysis of lactate dehydrogenase (LDH) sequences from thermophilic, mesophilic and psychrophilic bacilli revealed characteristic primary structural differences. These specific amino-acid substitutions were found in the entire LDH molecule. However, in certain regions of the LDH an accumulation of these exchanges could be detected. These regions seem to be particularly important for the temperature adaptation of the enzyme. The influence of one of such regions at the N-terminus on stability and activity of LDHs was analysed by the construction of hybrid mutants between LDH sequences from thermophilic, mesophilic and psychrophilic bacilli and also by site-directed mutagenesis experiments at five different positions. The substitutions of Thr-29 or Ser-39 to Ala residues in the LDH from the mesophilic B. megaterium increased the thermostability of the enzyme drastically (15 degrees C). An increase of 20 degrees C could be observed when both amino-acid substitutions were introduced. These amino-acid substitutions resulted in an increase of Km for pyruvate and led to a three-fold reduction of the activity (kcat/Km) at 40 degrees C compared with the wild type enzyme. The influence of these amino-acid substitutions was also investigated in the LDHs from thermophilic and psychrophilic bacilli. The high heat resistance of the LDH from the thermophilic B. stearothermophilus was not altered by the Ala to Thr and Ser substitutions at positions 29 and 39, respectively. This indicates a cooperatively stabilized conformation of this LDH. However, in this mutant of the B. stearothermophilus LDH the activity (kcat/Km) was increased two-fold.     

A catalytic mechanism that explains a low catalytic activity of serine dehydratase like-1 from human cancer cells: crystal structure and site-directed mutagenesis studies

SDH (l-serine dehydratase, EC 4.3.1.17) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes dehydration of l-Ser/Thr to yield pyruvate/ketobutyrate and ammonia. A SDH isoform (cSDH) found in human cancer cell lines has relatively low catalytic activity in comparison with the liver enzyme (hSDH). The crystal structure of cSDH has been determined at 2.8 angstroms resolution. A PLP is covalently attached to K48 by Schiff-base linkage in the active site. The ring nitrogen of PLP is involved in a H-bonding with C309, but is apparently not protonated. Twenty-three amino residues that compose the active site surfaces were identified. The human and rat liver enzymes (hSDH and rSDH) have the same residues, while residues G72, A172, and S228 in cSDH are replaced with A66, S166, and A222, respectively, in hSDH. These residues in hSDH and cSDH were mutated to make complementary pairs of mutated enzymes, and their kinetic parameters were determined. C303 of hSDH and C309 of cSDH which are H-bonding partner of the ring nitrogen of PLP were mutated to alanine and their kinetic parameters were also determined. The crystal structures and the mutation data suggest that having a glycine at residue 72 of cSDH is the major reason for the reduction of catalytic activity of cSDH. Changing alanine to glycine at residue 72 increases the flexibility of the substrate binding-loop (71S(G/A)GN74), so that the bound substrate and PLP are not pushed deep into the active cleft. Consequently, the proton transfer rate from S(G) of C309 to N1 of the bound PLP is decreased, which determines the rate of catalytic reaction.