Enzymatic activity of 2'-5'-oligoadenylate synthetase is impaired by specific mutations that affect oligomerization of the protein

Previous studies from our laboratory have shown that deletion of residues 321 to 344 of the 9-2 isozyme of 2'-5'-oligoadenylate (2-5(A)) synthetase causes a loss of its enzyme activity (Ghosh, S. K., Kusari, J., Bandyopadhyay, S. K., Samanta, H., Kumar, R., and Sen, G. C. (1991) J. Biol. Chem. 266, 15293-15299). Sequence comparison of this region among the different isozymes of 2-5(A) synthetases revealed that the residues at positions 330 to 333 are highly conserved. Alanine-scanning mutagenesis of these residues demonstrated that the residues present at 331, 332, and 333 are important for activity but the proline at position 330 was dispensable. The triple mutant containing Ala residues at 331, 332, and 333 was completely inactive. Different double mutants were slightly active, and the three single mutants were partially active. The triple mutant was further characterized for delineating the nature of its defect. The mutant protein was enzymatically inactive irrespective of whether it was synthesized in rabbit reticulocyte lysate, Escherichia coli or Trichoplusia ni insect cells. It could bind double-stranded RNA and ATP as efficiently as the wild type protein. It was, however, defective in oligomerization. Gel filtration and sedimentation velocity analyses of in vitro synthesized proteins revealed that the wild type protein, but not the triple mutant, formed tetramers. The tetrameric fraction, but not the monomeric fraction of the wild type protein was enzymatically active. The failure of the triple mutant to participate in homomeric protein-protein interaction was confirmed by in vivo assays in insect cells. These results indicate that tetramerization of the protein is required for the enzymatic activity of the small 2-5(A) synthetases.     

Production and purification of recombinant 2'-5' oligoadenylate synthetase and its mutants using the baculovirus system

Investigation of the structure-function relationship of the 2'-5' oligoadenylate [2-5 (A)] synthetases has been hampered by the lack of an efficient expression system for a recombinant enzyme. Here, we report that the 9-2 isozyme of murine 2-5 (A) synthetase can be efficiently expressed in insect cells using the baculovirus system. The recombinant protein was purified to apparent homogeneity, and its enzymatic activity was characterized. It had a high specific activity, required double-stranded RNA as a cofactor, and synthesized dimers to hexamers of 2-5 (A). The utility of our expression system was demonstrated by studying the properties of two previously reported mutant proteins. Both of these mutants, when produced in bacteria, are enzymatically inactive, although similarly produced wild-type protein is active. Unexpectedly, when expressed in insect cells, both mutant proteins were enzymatically as active as the wild-type protein. These results suggest that in the eukaryotic expression system described here, the mutant proteins can undergo appropriate modifications or folding that is required for attaining an enzymatically active conformation.     

Effect of cleavage mutants on syncytium formation directed by the wild-type fusion protein of Newcastle disease virus

The effects of Newcastle disease virus (NDV) fusion (F) glycoprotein cleavage mutants on the cleavage and syncytium-forming activity of the wild-type F protein were examined. F protein cleavage mutants were made by altering amino acids in the furin recognition region (amino acids 112 to 116) in the F protein of a virulent strain of NDV. Four mutants were made: Q114P replaced the glutamine residue with proline; K115G replaced lysine with glycine; double mutant K115G, R113G replaced both a lysine and an arginine with glycine residues; and a triple mutant, R112G, K115G, F117L, replaced three amino acids to mimic the sequence found in avirulent strains of NDV. All mutants except Q114P were cleavage negative and fusion negative. However, addition of exogenous trypsin cleaved all mutant F proteins and activated fusion. As expected for an oligomeric protein, the fusion-negative mutants had a dominant negative phenotype: cotransfection of wild-type and mutant F protein cDNAs resulted in an inhibition of syncytium formation. The presence of the mutant F protein did not inhibit cleavage of the wild-type protein. Furthermore, evidence is presented that suggests that the mutant protein and the wild-type protein formed heterooligomers. By measuring the syncytium-forming activity of the wild-type protein at various ratios of expression of mutant and wild-type protein, results were obtained that are most consistent with the notion that the size of the functionally active NDV F protein in these assays is a single oligomer, likely a trimer. That a larger oligomer, containing a mix of both wild-type and mutant F proteins, has partial activity cannot, however, be ruled out.     

Probing the role of cysteine residues in the EcoP15I DNA methyltransferase

Chemical modification using thiol-directed agents and site-directed mutagenesis has been used to investigate the role of cysteine residues of EcoP15I DNA methyltransferase. Irreversible inhibition of enzymatic activity was provoked by chemical modification of the enzyme by N-ethylmaleimide and iodoacetamide. 5, 5'-Dithiobis(2-nitrobenzoic acid) titration of the enzyme under nondenaturing and denaturing conditions confirmed the presence of six cysteine residues without any disulfides in the protein. Aware that relatively bulky reagents inactivate the methyltransferase by directly occluding the substrate-binding site or by locking the methyltransferase in an inactive conformation, we used site-directed mutagenesis to sequentially replace each of the six cysteines in the protein at positions 30, 213, 344, 434, 553, and 577. All the resultant mutant methylases except for the C344S and C344A enzymes retained significant activity as assessed by in vivo and in vitro assays. The effects of the substitutions on the function of EcoP15I DNA methyltransferase were investigated by substrate binding assays, activity measurements, and steady-state kinetic analysis of catalysis. Our results clearly indicate that the cysteines at positions other than 344 are not essential for activity. In contrast, the C344A enzyme showed a marked loss of enzymatic activity. More importantly, whereas the inactive C344A mutant enzyme bound S-adenosyl-L-methionine, it failed to bind to DNA. Furthermore, in double and triple mutants where two or three cysteine residues were replaced by serine, all such mutants in which the cysteine at position 344 was changed, were inactive. Taken together, these results convincingly demonstrate that the Cys-344 is necessary for enzyme activity and indicate an essential role for it in DNA binding.     

A conserved histidine is essential for glycerolipid acyltransferase catalysis

Sequence analysis of membrane-bound glycerolipid acyltransferases revealed that proteins from the bacterial, plant, and animal kingdoms share a highly conserved domain containing invariant histidine and aspartic acid residues separated by four less conserved residues in an HX4D configuration. We investigated the role of the invariant histidine residue in acyltransferase catalysis by site-directed mutagenesis of two representative members of this family, the sn-glycerol-3-phosphate acyltransferase (PlsB) and the bifunctional 2-acyl-glycerophosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase (Aas) of Escherichia coli. Both the PlsB[H306A] and Aas[H36A] mutants lacked acyltransferase activity. However, the Aas[H36A] mutant retained significant acyl-acyl carrier protein synthetase activity, illustrating that the lack of acyltransferase activity was specifically associated with the H36A substitution. The invariant aspartic acid residue in the HX4D pattern was also important. The substitution of aspartic acid 311 with glutamic acid in PlsB resulted in an enzyme with significantly reduced catalytic activity. Substitution of an alanine at this position eliminated acyltransferase activity; however, the PlsB[D311A] mutant protein did not assemble into the membrane, indicating that aspartic acid 311 is also important for the proper folding and membrane insertion of the acyltransferases. These data are consistent with a mechanism for glycerolipid acyltransferase catalysis where the invariant histidine functions as a general base to deprotonate the hydroxyl moiety of the acyl acceptor.     

Solution structure of P01, a natural scorpion peptide structurally analogous to scorpion toxins specific for apamin-sensitive potassium channel

The venom of the North African scorpion Androctonus mauretanicus mauretanicus possesses numerous highly active neurotoxins that specifically bind to various ion channels. One of these, P05, has been found to bind specifically to calcium-activated potassium channels and also to compete with apamin, a toxin extracted from bee venom. Besides the highly potent ones, several of these peptides (including that of P01) have been purified and been found to possess only a very weak, although significant, activity in competition with apamin. The amino acid sequence of P01 shows that it is shorter than P05 by two residues. This deletion occurs within an alpha-helix stretch (residues 5-12). This alpha-helix has been shown to be involved in the interaction of P05 with its receptor via two arginine residues. These two arginines are absent in the P01 sequence. Furthermore, a proline residue in position 7 of the P01 sequence may act as an alpha-helix breaker. We have determined the solution structure of P01 by conventional two-dimensional 1H nuclear magnetic resonance and show that 1) the proline residue does not disturb the alpha-helix running from residues 5 to 12; 2) the two arginines are topologically replaced by two acidic residues, which explains the drop in activity; 3) the residual binding activity may be due to the histidine residue in position 9; and 4) the overall secondary structure is conserved, i.e., an alpha-helix running from residues 5 to 12, two antiparallel stretches of beta-sheet (residues 15-20 and 23-27) connected by a type I' beta-turn, and three disulfide bridges connecting the alpha-helix to the beta-sheet.     

Enzymatic characteristics of recombinant medium isozyme of 2'-5' oligoadenylate synthetase

P69 is an isozyme of the medium size class of human 2'-5' oligoadenylate synthetases. In this study, recombinant P69 was expressed and used for enzymological and structural investigations. Bacterially expressed P69 was inactive whereas the same protein expressed in insect cells was highly active. Whether this difference could be due to differential post-translational modifications of the protein was investigated. Mutations of appropriate residues showed that myristoylation of the protein was not necessary for enzyme activity. In contrast, inhibition of glycosylation of P69, by tunicamycin treatment of the insect cells, produced an enzymatically inactive protein. Recombinant P69 produced in insect cells was purified by affinity chromatography. It was a dimeric glycoprotein, very stable and completely dependent on double stranded (ds) RNA for activity. The enzyme catalyzed the non-processive synthesis of 2'-5'-linked oligoadenylate products containing up to 30 residues. 2'-O-Methylated dsRNA was incapable of activating P69 and a 25-base pair dsRNA was as effective as larger dsRNA. This expression system will be useful for large scale production of P69 and its mutants for structural studies.     

Vitamin K2 (menaquinone) biosynthesis in Escherichia coli: evidence for the presence of an essential histidine residue in o-succinylbenzoyl coenzyme A synthetase

o-Succinylbenzoyl coenzyme A (OSB-CoA) synthetase, when treated with diethylpyrocarbonate (DEP), showed a time-dependent loss of enzyme activity. The inactivation follows pseudo-first-order kinetics with a second-order rate constant of 9.2 x 10(-4) +/- 1.4 x 10(-4) microM(-1) min(-1). The difference spectrum of the modified enzyme versus the native enzyme showed an increase in A242 that is characteristic of N-carbethoxyhistidine and was reversed by treatment with hydroxylamine. Inactivation due to nonspecific secondary structural changes in the protein and modification of tyrosine, lysine, or cysteine residues was ruled out. Kinetics of enzyme inactivation and the stoichiometry of histidine modification indicate that of the eight histidine residues modified per subunit of the enzyme, a single residue is responsible for the enzyme activity. A plot of the log reciprocal of the half-time of inactivation against the log DEP concentration further suggests that one histidine residue is involved in the catalysis. Further, the enzyme was partially protected from inactivation by either o-succinylbenzoic acid (OSB), ATP, or ATP plus Mg2+ while inactivation was completely prevented by the presence of the combination of OSB, ATP, and Mg2+. Thus, it appears that a histidine residue located at or near the active site of the enzyme is essential for activity. When His341 present in the previously identified ATP binding motif was mutated to Ala, the enzyme lost 65% of its activity and the Km for ATP increased 5.4-fold. Thus, His341 of OSB-CoA synthetase plays an important role in catalysis since it is probably involved in the binding of ATP to the enzyme.     

Mutagenesis and chemical rescue indicate residues involved in beta-aspartyl-AMP formation by Escherichia coli asparagine synthetase B

Site-directed mutagenesis and kinetic studies have been employed to identify amino acid residues involved in aspartate binding and transition state stabilization during the formation of beta-aspartyl-AMP in the reaction mechanism of Escherichia coli asparagine synthetase B (AS-B). Three conserved amino acids in the segment defined by residues 317-330 appear particularly crucial for enzymatic activity. For example, when Arg-325 is replaced by alanine or lysine, the resulting mutant enzymes possess no detectable asparagine synthetase activity. The catalytic activity of the R325A AS-B mutant can, however, be restored to about 1/6 of that of wild-type AS-B by the addition of guanidinium HCl (GdmHCl). Detailed kinetic analysis of the rescued activity suggests that Arg-325 is involved in stabilization of a pentacovalent intermediate leading to the formation beta-aspartyl-AMP. This rescue experiment is the second example in which the function of a critical arginine residue that has been substituted by mutagenesis is restored by GdmHCl. Mutation of Thr-322 and Thr-323 also produces enzymes with altered kinetic properties, suggesting that these threonines are involved in aspartate binding and/or stabilization of intermediates en route to beta-aspartyl-AMP. These experiments are the first to identify residues outside of the N-terminal glutamine amide transfer domain that have any functional role in asparagine synthesis.     

Affinity labeling of rat liver carbamyl phosphate synthetase I by 5'-p-fluorosulfonylbenzoyladenosine

The ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA) has been used to study the interaction of MgATP with rat liver carbamyl phosphate synthetase I. Incubation of the enzyme with concentrations of FSBA as low as 0.025 mM produced considerable inactivation (41% at 120 min); identical rates and extents of reaction were produced by 0.5, 1, and 2 mM FSBA. Of the substrates for carbamyl phosphate synthetase I, only MgATP protected against FSBA inactivation. In the presence of a constant concentration of MgATP, increasing the FSBA concentration led to increased inhibition. Conversely, an increase in MgATP concentration led to decreased inhibition from a constant concentration of FSBA. Other nucleotide triphosphates provided no protection against FSBA inactivation. Addition of dithiothreitol to the FSBA-inactivated enzyme led to partial reactivation, suggesting that cysteine residue(s) were involved in the FSBA reaction. 5,5'-Dithiobis(2-nitrobenzoic acid) titration of the free sulfhydryl groups on the enzyme confirmed that cysteine residues were involved in reaction with FSBA; titration of the enzyme after incubation in the absence and presence of FSBA yielded values of 21 and 18(+/- 1), respectively. Binding studies with 5'-p-fluorosulfonylbenzoyl[2-3H]adenosine indicated that: 4 amino acid residues were involved in reaction with FSBA; 2 of these reaction sites were cysteine residues and 2 were noncysteine residues; MgATP protected one of the cysteine residues and one of the noncysteine residues from reaction with FSBA; the MgATP-protected noncysteine residue is essential for fully activity. These data strongly suggest that FSBA is an affinity label for two distinct MgATP sites on carbamyl phosphate synthetase I.     

Site-directed mutagenesis of the yeast V-ATPase A subunit

To investigate the function of residues at the catalytic nucleotide binding site of the V-ATPase, we have carried out site-directed mutagenesis of the VMA1 gene encoding the A subunit of the V-ATPase in yeast. Of the three cysteine residues that are conserved in all A subunits sequenced thus far, two (Cys284 and Cys539) appear essential for correct folding or stability of the A subunit. Mutation of the third cysteine (Cys261), located in the glycine-rich loop, to valine, generated an enzyme that was fully active but resistant to inhibition by N-ethylmalemide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and oxidation. To test the role of disulfide bond formation in regulation of vacuolar acidification in vivo, we have also determined the effect of the C261V mutant on targeting and processing of the soluble vacuolar protein carboxypeptidase Y. No difference in carboxypeptidase Y targeting or processing is observed between the wild type and C261V mutant, suggesting that disulfide bond formation in the V-ATPase A subunit is not essential for controlling vacuolar acidification in the Golgi. In addition, fluid phase endocytosis of Lucifer Yellow, quinacrine staining of acidic intracellular compartments and cell growth are indistinguishable in the C261V and wild type cells. Mutation of G250D in the glycine-rich loop also resulted in destabilization of the A subunit, whereas mutation of the lysine residue in this region (K263Q) gave a V-ATPase complex which showed normal levels of A subunit on the vacuolar membrane but was unstable to detergent solubilization and isolation and was totally lacking in V-ATPase activity. By contrast, mutation of the acidic residue, which has been postulated to play a direct catalytic role in the homologous F-ATPases (E286Q), had no effect on stability or assembly of the V-ATPase complex, but also led to complete loss of V-ATPase activity. The E286Q mutant showed labeling by 2-azido-[32P]ATP that was approximately 60% of that observed for wild type, suggesting that mutation of this glutamic acid residue affected primarily ATP hydrolysis rather than nucleotide binding.     

Characterization of the catalytic site of the ADP-ribosyltransferase Clostridium botulinum C2 toxin by site-directed mutagenesis

The actin ADP-ribosylating Clostridium botulinum C2 toxin is a binary toxin composed of the binding component C2II and the enzyme component C2I. C2I ADP-ribosylates G-actin at arginine 177, resulting in the depolymerization of the actin cytoskeleton. Here, we studied the structure-function relationship of C2I by site-directed mutagenesis. Exchange of Glu389 to glutamine caused the complete loss of ADP-ribosyltransferase and NAD-glycohydrolase activities of C2I. In contrast, exchange of Glu387 to glutamine blocked ADP-ribosyltransferase but not NAD-glycohydrolase activity. Whereas photoaffinity labeling of the double mutant E387Q/E389Q C2I with [carbonyl-14C]NAD was blocked, labeling of the single C2I mutants was reduced (E389Q) or not changed (E387Q). Exchange of the STS motif (amino acid residues 348-350) of C2I caused a decrease in transferase activity by more than 99 (S348A) and 90% (T349V), or did not affect activity (S350A). Exchange of Arg299 and Arg300 to lysine reduced transferase activity to <0.1 and approximately 35% of wild-type activity. The data indicate that the amino acid residues Glu389, Glu387, Ser348, and Arg299, which are conserved in various prokaryotic and eukaryotic arginine-modifying ADP-ribosyltransferases, are essential for ADP-ribosyltransferase activity of the enzyme component of C. botulinum C2 toxin.     

Combined spinal-epidural anesthesia for outpatient surgery. Dose-response characteristics of intrathecal isobaric lidocaine using a 27-gauge Whitacre spinal needle

Leukotriene A4 (LTA4) hydrolase is a bifunctional zinc metalloenzyme which catalyzes the final step in the biosynthesis of the proinflammatory leukotriene B4 and which also possesses a peptidase activity. From sequence comparisons with aminopeptidases, a tyrosine at position 383 in LTA4 hydrolase has been suggested as a possible catalytic amino acid. To explore the potential role of this amino acid in catalysis, we replaced the tyrosine residue with phenylalanine, histidine or glutamine residues by site-directed mutagenesis. The mutated cDNAs were expressed in Escherichia coli and the resulting recombinant proteins, named [Y383F]LTA4 hydrolase, [Y383H]LTA4 hydrolase and [Y383Q]LTA4 hydrolase, were purified to homogeneity to allow assays of both the epoxide hydrolase activity, i.e. the conversion of LTA4 into leukotriene B4, and the peptidase activity. None of the mutated proteins exhibited significant peptidase activities, all of them showing activities less than 0.3% that of the wild-type enzyme. The epoxide hydrolase activity was not affected to the same degree and corresponded to 11, 16 and 17% that of the unmutated enzyme for [Y383F]LTA4 hydrolase, [Y383H]LTA4 hydrolase and [Y383Q]LTA4 hydrolase, respectively. Kinetic analysis was performed with the mutant [Y383Q]LTA4 hydrolase, which revealed an approximately 10-fold increase in Km for leukotriene A4 compared to that for the unmutated enzyme. At high concentrations of substrate, the difference in enzyme velocity was only moderate, with Vmax values of 600 nmol.mg-1.min-1 and 1000 nmol.mg-1.min-1 for [Y383Q]LTA4 hydrolase and the wild-type enzyme, respectively. No such effect of substrate concentration could be observed on the peptidase activity. As a positive control, we exchanged a glycine residue in position 386 for an alanine residue, and the recombinant protein, [G386A]LTA4 hydrolase retained 19% and 77% of the peptidase and epoxide hydrolase activities, respectively. The results from this study are consistent with a role for Tyr383 in the peptidase reaction of LTA4 hydrolase, where it may act as a proton donor in a general base mechanism. However, our data do not allow a similar interpretation for the mechanism involved in the hydrolysis of LTA4 into LTB4.     

Site-directed mutagenesis of residues in a conserved region of bovine aspartyl (asparaginyl) beta-hydroxylase: evidence that histidine 675 has a role in binding Fe2+

The roles in catalysis of several residues in bovine aspartyl (asparaginyl) beta-hydroxylase that are located in a region of homology among alpha-ketoglutarate-dependent dioxygenases were investigated using site-directed mutagenesis. Previous studies have shown that when histidine 675, an invariant residue located in this highly conserved region, was mutated to an alanine residue, no enzymatic activity was detected. A more extensive site-directed mutagenesis study at position 675 has been undertaken to define the catalytic role of this essential residue. The partial hydroxylase activity observed with some amino acid replacements for histidine 675 correlates with the potential to coordinate metals and not with size, charge, or hydrophobic character. Furthermore, the increase in Km for Fe2+ observed with the H675D and H675E mutant enzymes can account for their partial activities relative to wild type. No significant changes in the Km for alpha-ketoglutarate (at saturating Fe2+) or Vmax were observed for these mutants. These results support the conclusion that histidine 675 is specifically involved in Fe2+ coordination. Further site-directed mutagenesis of other highly conserved residues in the vicinity of position 675 demonstrates the importance of this region of homology in catalysis for Asp (Asn) beta-hydroxylase and, by analogy, other alpha-ketoglutarate-dependent dioxygenases.     

Site-directed mutagenesis studies of the metal-binding center of the iron-dependent propanediol oxidoreductase from Escherichia coli

The amino acid residues involved in the metal-binding site in the iron-containing dehydrogenase family were characterized by the site-directed mutagenesis of selected candidate residues of propanediol oxidoreductase from Escherichia coli. Based on the findings that mutations H263R, H267A and H277A resulted in iron-deficient propanediol oxidoreductases without catalytic activity, we identified three conserved His residues as iron ligands, which also bind zinc. The Cys362, a residue highly conserved among these dehydrogenases, was considered another possible ligand by comparison with the sequences of the medium-chain dehydrogenases. Mutation of Cys362 to Ile, resulted in an active enzyme that was still able to bind iron, with minor changes in the Km values and decreased thermal stability. Furthermore, in an attempt to produce an enzyme specific only for the zinc ion, three mutations were designed to mimic the catalytic zinc-binding site of the medium-chain dehydrogenases: (1) V262C produced an enzyme with altered kinetic parameters which nevertheless retained a significant ability to bind both metals, (2) the double mutant V262C-M265D was inactive and too unstable to allow purification, and (3) the insertion of a cysteine at position 263 resulted in a catalytically inactive enzyme without iron-binding capacity, while retaining the ability to bind zinc. This mutation could represent a conceivable model of one of the steps in the evolution from iron to zinc-dependent dehydrogenases.     

Mutational analysis of potential zinc-binding residues in the active site of the enterococcal D-Ala-D-Ala dipeptidase VanX

VanX, one of the five proteins required for the vancomycin-resistant phenotype in clinically pathogenic Enterococci, is a zinc-containing d-Ala-d-Ala dipeptidase. To identify potential zinc ligands and begin defining the active site residues, we have mutated the 2 cysteine, 5 histidine, and 4 of the 28 aspartate and glutamate residues in the 202 residue VanX protein. Of 10 mutations, 3 cause inactivation and greater than 90% loss of zinc in purified enzyme samples, implicating His116, Asp123, and His184 as zinc-coordinating residues. Homology searches using the 10 amino acid sequence SxHxxGxAxD, in which histidine and aspartate residues are putative zinc ligands, identified the metal coordinating ligands in the N-terminal domain of the murine Sonic hedgehog protein, which also exhibits an architecture for metal coordination identical to that observed in thermolysin from Bacillus thermoproteolyticus. Furthermore, this 10 amino acid consensus sequence is found in the Streptomyces albus G zinc-dependent N-acyl-d-Ala-d-Ala carboxypeptidase, an enzyme catalyzing essentially the same d-Ala-d-Ala dipeptide bond cleavage as VanX, suggesting equivalent mechanisms and zinc catalytic site architectures. VanX residue Glu181 is analogous to the Glu143 catalytic base in B. thermoproteolyticus thermolysin, and the E181A VanX mutant has no detectable dipeptidase activity, yet maintains near-stoichiometric zinc content, a result consistent with the participation of the residue as a catalytic base.     

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.