Purification,characterization, and molecular cloning of a novel amine:pyruvate transaminase from Vibrio fluvialis JS17

A transaminase from Vibrio fluvialis JS17 showing activity toward chiral amines was purified to homogeneity and its enzymatic properties were characterized. The transaminase showed an apparent molecular mass of 100 kDa as determined by gel filtration chromatography and a subunit mass of 50 kDa by MALDI-TOF mass spectrometry, suggesting a dimeric structure. The enzyme had an isoelectric point of 5.4 and its absorption spectrum exhibited maxima at 320 and 405 nm. The optimal pH and temperature for enzyme activity were 9.2 and 37 degrees C, respectively. Pyruvate and pyridoxal 5'-phosphate increased enzyme stability whereas (S)-alpha-methylbenzylamine reversibly inactivated the enzyme. The transaminase gene was cloned from a V. fluvialis JS17 genomic library. The deduced amino acid sequence (453 residues) showed significant homology with omega-amino acid:pyruvate transaminases (omega-APT) from various bacterial strains (80 identical residues with four omega-APTs). However, of 159 conserved residues in the four omega-APTs, 79 were not conserved in the transaminase from V. fluvialis JS17. Taken together with the sequence homology results, and the lack of activity toward beta-alanine (a typical amino donor for the omega-APT), the results suggest that the transaminase is a novel amine:pyruvate transaminase that has not been reported to date.     

The primary structure of thermostable D-amino acid aminotransferase from a thermophilic Bacillus species and its correlation with L-amino acid aminotransferases

The gene for thermostable D-amino acid aminotransferase from a thermophile, Bacillus species YM-1 was cloned and expressed efficiently in Escherichia coli. The entire covalent structure of the enzyme was determined from the nucleotide sequence of the cloned gene and mostly confirmed by amino acid sequences of tryptic peptides from the gene product. The polypeptide is composed of 282 amino acid residues with a calculated molecular weight of 32,226. Comparison of the primary structure with those of various proteins registered in a protein data bank revealed a significant sequence homology between D-amino acid aminotransferase and the L-branched chain amino acid aminotransferase of E. coli (Kuramitsu, S., Ogawa, T., Ogawa, H., and Kagamiyama, H. (1985) J. Biochem. (Tokyo) 97, 993-999); the active site lysyl residue is located in an equivalent position in both enzyme sequences of similar size. Despite the difference in subunit composition and no immunochemical cross-reactivity, the sequences of the two enzymes show similar hydropathy profiles, and spectrophotometric properties of the enzyme-bound cofactor are also similar. The sequence homology suggests that the structural genes for D-amino acid and L-branched chain amino acid aminotransferases evolved from a common ancestral gene.     

Complete amino acid sequence of rat liver cytosolic alanine aminotransferase

The complete amino acid sequence of rat liver cytosolic alanine aminotransferase (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The molecular weight of the subunit was calculated to be 55,018 which was in good agreement with a molecular weight of 55,000 determined by SDS-PAGE and also indicated that the active enzyme with a molecular weight of 114,000 was a homodimer composed of two identical subunits. No highly homologous sequence was found in protein sequence databases except for a 20-residue sequence around the pyridoxal 5'-phosphate binding site of the pig heart enzyme [Tanase, S., Kojima, H., & Morino, Y. (1979) Biochemistry 18, 3002-3007], which was almost identical with that of residues 303-322 of the rat liver enzyme. In spite of rather low homology scores, rat alanine aminotransferase is clearly homologous to those of other aminotransferases from the same species, e.g., cytosolic tyrosine aminotransferase (24.7% identity), cytosolic aspartate aminotransferase (17.0%), and mitochondrial aspartate aminotransferase (16.0%). Most of the crucial amino acid residues hydrogen-bonding to pyridoxal 5'-phosphate identified in aspartate aminotransferase by X-ray crystallography are conserved in alanine aminotransferase. This suggests that the topology of secondary structures characteristic in the large domain of other alpha-aminotransferases with known tertiary structure may also be conserved in alanine aminotransferase.     

Cloning and sequence analysis of mRNA for mouse aspartate aminotransferase isoenzymes

The nucleotide sequences of mRNAs for the mouse mitochondrial and cytosolic aspartate aminotransferase isoenzymes (mAspAT and cAspAT) (EC 2.6.1.1) were determined from complementary DNAs. The mAspAT mRNA comprises minimally 2460 nucleotides and codes for a polypeptide of 430 amino acid residues corresponding to the precursor form of the mAspAT (pre-mAspAT). The cAspAT mRNA comprises minimally 2086 nucleotides and codes for a polypeptide of 413 amino acid residues. The region coding for the mature mAspAT and that for the cAspAT show about 53% overall homology. The former shares 49% and the latter 48% of homology, respectively, with that of the Escherichia coli aspC gene, which has been shown to code for the E. coli AspAT (Kuramitsu, S., Okuno, S., Ogawa, T., Ogawa, H., and Kagamiyama, H. (1985) J. Biochem. (Tokyo) 97, 1259-1262). When the deduced amino acid sequence of the mouse pre-mAspAT was compared with that of the pig pre-mAspAT polypeptide, we found that they share a 94% homology and that the mouse pre-mAspAT yields a presequence consisting of 29 amino acid residues and a mature mAspAT, consisting of 401 amino acid residues. These numbers and the amino acid residues present at the putative cleavage site are all in complete agreement in these two species. The deduced amino acid sequence of the mouse cAspAT shares 91% homology with that of the pig cAspAT. Comparisons of the nucleotide and deduced amino acid sequences between the mouse and E. coli AspATs suggest that the mammalian mAspAT gene is more closely related to the E. coli aspC gene than is the mammalian cAspAT gene.     

Characterization of aspartate aminotransferase from the cyanobacterium Phormidium lapideum

Aspartate aminotransferase (AspAT) was purified to homogeneity from cell extracts of the non-N2-fixing cyanobacterium Phormidium lapideum. The NH2-terminal sequence of 25 amino acid residues was different from the sequences of the subfamily Ialpha of AspATs from eukaryotes and Escherichia coli, but it was similar to sequences of the subfamily Igamma of AspATs from archaebacteria and eubacteria. The enzyme was most active at 80 degrees C and was stable at up to 75 degrees C. Thermal inactivation (60-85 degrees C) of the enzyme followed first-order kinetics, with 2-oxoglutarate causing a shift of the thermal inactivation curves to higher temperatures. However, at 25 degrees C the kcat of P. lapideum AspAT was nearly equal to the values of AspATs from mesophilic organisms. The enzyme used L-aspartate and L-cysteine sulfinate as amino donors and 2-oxoglutarate as an amino acceptor. The Km values were 5.0 mM for L-aspartate, 5.7 mM for L-glutamate, 0.2 mM for 2-oxoglutarate, and 0.032 mM for oxaloacetate.     

Stereochemistry of the transamination reaction catalyzed by aminodeoxychorismate lyase from Escherichia coli: close relationship between fold type and stereochemistry

Aminodeoxychorismate lyase is a pyridoxal 5'-phosphate-dependent enzyme that converts 4-aminodeoxychorismate to pyruvate and p-aminobenzoate, a precursor of folic acid in bacteria. The enzyme exhibits significant sequence similarity to two aminotransferases, D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase. In the present study, we have found that aminodeoxychorismate lyase catalyzes the transamination between D-alanine and pyridoxal phosphate to produce pyruvate and pyridoxamine phosphate. L-Alanine and other D- and L-amino acids tested were inert as substrates of transamination. The pro-R hydrogen of C4' of pyridoxamine phosphate was stereospecifically abstracted during the reverse half transamination from pyridoxamine phosphate to pyruvate. Aminodeoxychorismate lyase is identical to D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase in the stereospecificity of the hydrogen abstraction, and differs from all other pyridoxal enzymes that catalyze pro-S hydrogen transfer. Aminodeoxychorismate lyase is the first example of a lyase that catalyzes pro-R-specific hydrogen abstraction. The result is consistent with recent X-ray crystallographic findings showing that the topological relationships between the cofactor and the catalytic residue for hydrogen abstraction are conserved among aminodeoxychorismate lyase, D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase [Nakai, T., Mizutani, H., Miyahara, I., Hirotsu, K., Takeda, S., Jhee, K.-H., Yoshimura, T., and Esaki, N. (2000) J. Biochem. 128, 29-38].     

Kinetic properties of mutant human thymidine kinase 2 suggest a mechanism for mitochondrial DNA depletion myopathy

Thymidine kinase 2 (TK2) is a mitochondrial (mt) pyrimidine deoxynucleoside salvage enzyme involved in mtDNA precursor synthesis. The full-length human TK2 cDNA was cloned and sequenced. A discrepancy at amino acid 37 within the mt leader sequence in the DNA compared with the determined peptide sequence was found. Two mutations in the human TK2 gene, His-121 to Asn and Ile-212 to Asn, were recently described in patients with severe mtDNA depletion myopathy (Saada, A., Shaag, A., Mandel, H., Nevo, Y., Eriksson, S., and Elpeleg, O. (2001) Nat. Genet. 29, 342-344). The same mutations in TK2 were introduced, and the mutant enzymes, prepared in recombinant form, were shown to have similar subunit structure to wild type TK2. The I212N mutant showed less than 1% activity as compared with wild type TK2 with all deoxynucleosides. The H121N mutant enzyme had normal K(m) values for thymidine (dThd) and deoxycytidine (dCyd), 6 and 11 microm, respectively, but 2- and 3-fold lower V(max) values as compared with wild type TK2 and markedly increased K(m) values for ATP, leading to decreased enzyme efficiency. Competition experiments revealed that dCyd and dThd interacted differently with the H121N mutant as compared with the wild type enzyme. The consequences of the two point mutations of TK2 and the role of TK2 in mt disorders are discussed.     

Crystal structure of Trypanosoma cruzi tyrosine aminotransferase: substrate specificity is influenced by cofactor binding mode

The crystal structure of tyrosine aminotransferase (TAT) from the parasitic protozoan Trypanosoma cruzi, which belongs to the aminotransferase subfamily Igamma, has been determined at 2.5 A resolution with the R-value R = 15.1%. T. cruzi TAT shares less than 15% sequence identity with aminotransferases of subfamily Ialpha but shows only two larger topological differences to the aspartate aminotransferases (AspATs). First, TAT contains a loop protruding from the enzyme surface in the larger cofactor-binding domain, where the AspATs have a kinked alpha-helix. Second, in the smaller substrate-binding domain, TAT has a four-stranded antiparallel beta-sheet instead of the two-stranded beta-sheet in the AspATs. The position of the aromatic ring of the pyridoxal-5'-phosphate cofactor is very similar to the AspATs but the phosphate group, in contrast, is closer to the substrate-binding site with one of its oxygen atoms pointing toward the substrate. Differences in substrate specificities of T. cruzi TAT and subfamily Ialpha aminotransferases can be attributed by modeling of substrate complexes mainly to this different position of the cofactor-phosphate group. Absence of the arginine, which in the AspATs fixes the substrate side-chain carboxylate group by a salt bridge, contributes to the inability of T. cruzi TAT to transaminate acidic amino acids. The preference of TAT for tyrosine is probably related to the ability of Asn17 in TAT to form a hydrogen bond to the tyrosine side-chain hydroxyl group.     

Complete amino acid sequence of human liver cytosolic alanine aminotransferase (GPT) determined by a combination of conventional and mass spectral methods

The complete amino acid sequence of human liver cytosolic alanine aminotransferase (GPT) (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. Isolated peptides were analyzed with a protein sequencer or with a plasma desorption time of flight mass spectrometer and placed in the sequence on the basis of their molecular mass and homology to the sequence of rat GPT. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The Mr of the subunit was calculated to be 54,479, which was in good agreement with a Mr of 55,000 estimated by SDS-PAGE, and also indicated that the active enzyme with a Mr of 114,000 was a homodimer composed of two identical subunits. The amino acid sequence is highly homologous to that of rat GPT (87.9% identity) recently determined [Ishiguro, M., Suzuki, M., Takio, K., Matsuzawa, T., & Titani, K. (1991) Biochemistry 30, 6048-6053]. All of the crucial amino acid residues are conserved in human GPT, which seem to be hydrogen bonding to pyridoxal 5'-phosphate in rat GPT by the sequence homology to other alpha-aminotransferases with known tertiary structures.     

Structures of Escherichia coli histidinol-phosphate aminotransferase and its complexes with histidinol-phosphate and N-(5'-phosphopyridoxyl)-L-glutamate: double substrate recognition of the enzyme

Histidinol-phosphate aminotransferase (HspAT) is a key enzyme on the histidine biosynthetic pathway. HspAT catalyzes the transfer of the amino group of L-histidinol phosphate (Hsp) to 2-oxoglutarate to form imidazole acetol phosphate (IAP) and glutamate. Thus, HspAT recognizes two kinds of substrates, Hsp and glutamate (double substrate recognition). The crystal structures of native HspAT and its complexes with Hsp and N-(5'-phosphopyridoxyl)-L-glutamate have been solved and refined to R-factors of 19.7, 19.1, and 17.8% at 2.0, 2.2, and 2.3 A resolution, respectively. The enzyme is a homodimer, and the polypeptide chain of the subunit is folded into one arm, one small domain, and one large domain. Aspartate aminotransferases (AspATs) from many species were classified into aminotransferase subgroups Ia and Ib. The primary sequence of HspAT is less than 18% identical to those of Escherichia coli AspAT of subgroup Ia and Thermus thermophilus HB8 AspAT of subgroup Ib. The X-ray analysis of HspAT showed that the overall structure is significantly similar to that of AspAT of subgroup Ib rather than subgroup Ia, and the N-terminal region moves close to the active site like that of subgroup Ib AspAT upon binding of Hsp. The folding of the main-chain atoms in the active site is conserved between HspAT and the AspATs, and more than 40% of the active-site residues is also conserved. The eHspAT recognizes both Hsp and glutamate by utilizing essentially the same active-site folding as that of AspAT, conserving the essential residues for transamination reaction, and replacing and relocating some of the active-site residues. The binding sites for the phosphate and the alpha-carboxylate groups of the substrates are roughly located at the same position and those for the imidazole and gamma-carboxylate groups at the different positions. The mechanism for the double substrate recognition observed in eHspAT is in contrast to that in aromatic amino acid aminotransferase, where the recognition site for the side chain of the acidic amino acid is formed at the same position as that for the side chain of aromatic amino acids by large-scale rearrangements of the hydrogen bond networks.     

Primary structure of copper-zinc superoxide dismutase from Neurospora crassa

The complete amino acid sequence of copper-zinc superoxide dismutase from Neurospora crassa is reported. The subunit consists of 153 amino acids and has a Mr of 15,850. The primary structure was determined by automated and manual sequence analysis of peptides obtained by digestions of the carboxymethylated and aminoethylated enzyme with trypsin and thermolysin. The protein is devoid of tryptophan and methionine and displays a free amino terminus. Comparison of the amino acid sequence with those from human erythrocyte, bovine erythrocyte, horse liver, swordfish liver, and yeast copper-zinc superoxide dismutases reveals a high degree of sequence homology among the six enzymes. Most prominently, the regions containing the amino acid residues participating in the metal-binding and the half-cystine residues forming the intramolecular disulfide bridge are highly conserved. The invariant amino acids Pro 74 and Asp 76 of the four vertebrate and yeast superoxide dismutases were found to be substituted by arginine and alanine, respectively, in the Neurospora enzyme. These radical substitutions occurring in the zinc ligand region, known to form a characteristic loop structure in bovine erythrocyte copper-zinc superoxide dismutase (Tainer, J. A., Getzoff, E. D., Beem, K. M., Richardson, J. S., and Richardson, D. C. (1982) J. Mol. Biol. 160, 181-217), however, do not affect the catalytic properties of the Neurospora enzyme.     

Structure of Thermus thermophilus HB8 aspartate aminotransferase and its complex with maleate

The three-dimensional structures of pyridoxal 5'-phosphate-type aspartate aminotransferase (AspAT) from Thermus thermophilus HB8 and pyridoxamine 5'-phosphate type one in complex with maleate have been determined by X-ray crystallography at 1.8 and 2.6 A resolution, respectively. The enzyme is a homodimer, and the polypeptide chain of the subunit is folded into one arm, one small domain, and one large domain. AspATs from many species were classified into aminotransferase subgroups Ia and Ib. The enzyme belongs to subgroup Ib, its sequence being less than 16% identical to the primary sequences of Escherichia coli, pig cytosolic, and chicken mitochondrial AspATs, which belong to subgroup Ia whose sequences are more than 40% identical and whose three-dimensional structures are quite similar with the active site residues almost completely conserved. The first X-ray analysis of AspAT subgroup Ib indicated that the overall and the active site structures are essentially conserved between the AspATs of subgroup Ia and the enzyme of subgroup Ib, but there are two distinct differences between them. (1) In AspAT subgroup Ia, substrate (or inhibitor) binding induces a large movement of the small domain as a whole to close the active site. However, in the enzyme of subgroup Ib, only the N-terminal region (Lys13-Val30) of the small domain approaches the active site to interact with the maleate. (2) In AspAT subgroup Ia, Arg292 recognizes the side chain carboxylate of the substrate; however, residue 292 of the enzyme in subgroup Ib is not Arg, and in place of Arg292, Lys109 forms a salt bridge with the side chain carboxylate. The thermostability of the enzyme is attained at least in part by the high content of Pro residues in the beta-turns and the marked increase in the number of salt bridges on the molecular surface compared with the mesophilic AspAT.     

Rat liver polysome N alpha-acetyltransferase: isolation and characterization

Rat liver polysome N alpha-acetyltransferase has been purified to homogeneity by a four-step procedure that utilizes ammonium sulfate precipitation, gel filtration, hydroxylapatite chromatography, and Mono Q ion exchange chromatography. The enzyme is greatly stabilized by the inclusion of EDTA and 0.01% deoxycholate in the isolation buffers. The purified enzyme has a native molecular weight of 190,000 and a subunit molecular weight of 95,000, suggesting that it is a homodimer. The enzyme shows a pH optimum of 8.0 and is strongly inhibited by Cl-, I-, SCN-, and ClO4- and to a lesser degree by sulfate and acetate. It is unaffected by phosphate, citrate, and F- and by Na+ and K+; NH4+ is partially inhibitory. The enzyme is also sensitive to iodoacetic acid. It is generally more similar to yeast N alpha-acetyltransferase [Lee, F.-J. S., Lin, L.-W., & Smith, J. A. (1988) J. Biol. Chem. 263, 14948-14955] than to the hen oviduct enzyme, which contains a 7S RNA subunit [Kamitani, K., & Sakiyama, F. (1989) J. Biol. Chem. 264, 13194-13198], although the amino acid compositions are quite different.     

The primary structure of aspartate aminotransferase from pig heart muscle. Partial sequences determined by digestion with thermolysin and elastase

Peptides produced by thermolytic digestion of aminoethylated aspartate aminotransferase and of the oxidized enzyme were isolated and their amino acid sequences determined. Digestion by elastase of the carboxymethylated enzyme gave peptides representing approximately 40% of the primary structure. Fragments from these digests overlapped with previously reported sequences of peptides obtained by peptic and tryptic digestion (Doonan et al., 1972), giving ten composite peptides containing 395 amino acid residues. The amino acid composition of these composite peptides agrees well with that of the intact enzyme. Confirmatory results for some of the present data have been deposited as Supplementary Publication 50018 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.     

Aspartate aminotransferase of Escherichia coli: nucleotide sequence of the aspC gene

The nucleotide sequence of the aspartate aminotransferase [EC 2.6.1.1] structural gene, aspC, of Escherichia coli K-12 was determined. The coding region of the aspC gene contained 1,188 nucleotide residues and encoded 396 amino acid residues. The amino acid sequence deduced from the nucleotide sequence agreed perfectly with that of the protein recently determined for the aspartate aminotransferase of E. coli B (Kondo, K., Wakabayashi, S., Yagi, T., & Kagamiyama, H. (1984) Biochem. Biophys. Res. Commun. 122, 62-67).     

Purification and properties of the aromatic amino acid aminotransferase from gramicidin S-producing Bacillus brevis

The aromatic amino acid aminotransferase was purified to a homogenous state from a gramicidin S-producing strain of Bacillus brevis. The enzyme shows a molecular weight of about 71,000 on gel-filtration. The subunit molecular weight is about 35,000 as determined by sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is a dimer. The enzyme exhibits absorption maxima near 425 and 330 nm at neutral pH. One mole of pyridoxal phosphate is bound per subunit. The enzyme has amino donor specificity for aromatic amino acids, L-phenylalanine, L-tyrosine, and L-tryptophan, and utilizes 2-oxoglutarate as the amino acceptor. This enzyme activity was separated from both the aspartate aminotransferase activity and the branched chain amino acid aminotransferase activity by chromatography on DEAE-Sephadex.     

Thermostable D-amino acid aminotransferase from a thermophilic Bacillus species. Purification, characterization, and active site sequence determination

D-Amino acid aminotransferase was found in several thermophilic Bacillus species and purified to homogeneity from the best producer, Bacillus sp. YM-1, which was newly isolated from a sauna dust. The enzyme has a molecular weight of about 62,000 and consists of two subunits identical in molecular weight (30,000). It catalyzes transamination between various D-amino acids and alpha-keto acids, although the substrate specificity is narrower than the enzyme from the mesophile, Bacillus sphaericus (Yonaha, K., Misono, H., Yamamoto, T., and Soda, K. (1975) J. Biol. Chem. 250, 6983-6989). The Bacillus sp. YM-1 enzyme is most active at 60 degrees C and stable at high temperatures. Automated Edman degradation provided the N-terminal sequence of the first 20 amino acids, and carboxypeptidase Y digestion provided the C-terminal sequence of the last 3 amino acids. The amino acid sequence in the vicinity of the lysyl residue, Lys(Pxy), that binds pyridoxal 5'-phosphate was determined as Cys-Asp-Ile-Lys(Pxy)-Ser-Leu-Asn-Leu-Leu-Gly-Ala-Val-Leu-Ala-Lys- from the pyridoxyl peptide obtained by digestion with trypsin. The active site sequence is markedly different from those of L-amino acid aminotransferases and other pyridoxal 5'-phosphate-dependent enzymes.     

Tyrosine aminotransferase catalyzes the final step of methionine recycling in Klebsiella pneumoniae

An aminotransferase which catalyzes the final step in methionine recycling from methylthioadenosine, the conversion of alpha-ketomethiobutyrate to methionine, has been purified from Klebsiella pneumoniae and characterized. The enzyme was found to be a homodimer of 45-kDa subunits, and it catalyzed methionine formation primarily using aromatic amino acids and glutamate as the amino donors. Histidine, leucine, asparagine, and arginine were also functional amino donors but to a lesser extent. The N-terminal amino acid sequence of the enzyme was determined and found to be almost identical to the N-terminal sequence of both the Escherichia coli and Salmonella typhimurium tyrosine aminotransferases (tyrB gene products). The structural gene for the tyrosine aminotransferase was cloned from K. pneumoniae and expressed in E. coli. The deduced amino acid sequence displayed 83, 80, 38, and 34% identity to the tyrosine aminotransferases from E. coli, S. typhimurium, Paracoccus denitrificans, and Rhizobium meliloti, respectively, but it showed less than 13% identity to any characterized eukaryotic tyrosine aminotransferase. Structural motifs around key invariant residues placed the K. pneumoniae enzyme within the Ia subfamily of aminotransferases. Kinetic analysis of the aminotransferase showed that reactions of an aromatic amino acid with alpha-ketomethiobutyrate and of glutamate with alpha-ketomethiobutyrate proceed as favorably as the well-known reactions of tyrosine with alpha-ketoglutarate and tyrosine with oxaloacetate normally associated with tyrosine aminotransferases. The aminotransferase was inhibited by the aminooxy compounds canaline and carboxymethoxylamine but not by substrate analogues, such as nitrotyrosine or nitrophenylalanine.     

Molecular cloning, expression and characterization of pyridoxamine-pyruvate aminotransferase

Pyridoxamine-pyruvate aminotransferase is a PLP (pyridoxal 5'-phosphate) (a coenzyme form of vitamin B6)-independent aminotransferase which catalyses a reversible transamination reaction between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The gene encoding the enzyme has been identified, cloned and overexpressed for the first time. The mlr6806 gene on the chromosome of a symbiotic nitrogen-fixing bacterium, Mesorhizobium loti, encoded the enzyme, which consists of 393 amino acid residues. The primary sequence was identical with those of archaeal aspartate aminotransferase and rat serine-pyruvate aminotransferase, which are PLP-dependent aminotransferases. The results of fold-type analysis and the consensus amino acid residues found around the active-site lysine residue identified in the present study showed that the enzyme could be classified into class V aminotransferases of fold type I or the AT IV subfamily of the alpha family of the PLP-dependent enzymes. Analyses of the absorption and CD spectra of the wild-type and point-mutated enzymes showed that Lys197 was essential for the enzyme activity, and was the active-site lysine residue that corresponded to that found in the PLP-dependent aminotransferases, as had been suggested previously [Hodsdon, Kolb, Snell and Cole (1978) Biochem. J. 169, 429-432]. The K(d) value for pyridoxal determined by means of CD was 100-fold lower than the K(m) value for it, suggesting that Schiff base formation between pyridoxal and the active-site lysine residue is partially rate determining in the catalysis of pyridoxal. The active-site structure and evolutionary aspects of the enzyme are discussed.     

Nucleotide sequence of the luxA gene of Vibrio harveyi and the complete amino acid sequence of the alpha subunit of bacterial luciferase

The nucleotide sequence of the 1.85-kilobase EcoRI fragment from Vibrio harveyi that was cloned using a mixed-sequence synthetic oligonucleotide probe (Cohn, D. H., Ogden, R. C., Abelson, J. N., Baldwin, T. O., Nealson, K. H., Simon, M. I., and Mileham, A. J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 120-123) has been determined. The alpha subunit-coding region (luxA) was found to begin at base number 707 and end at base number 1771. The alpha subunit has a calculated molecular weight of 40,108 and comprises a total of 355 amino acid residues. There are 34 base pairs separating the start of the alpha subunit structural gene and a 669-base open reading frame extending from the proximal EcoRI site. At the 3' end of the luxA coding region there are 26 bases between the end of the structural gene and the start of the luxB structural gene. Approximately two-thirds of the alpha subunit was sequenced by protein chemical techniques. The amino acid sequence implied by the DNA sequence, with few exceptions, confirmed the chemically determined sequence. Regions of the alpha subunit thought to comprise the active center were found to reside in two discrete and relatively basic regions, one from around residues 100-115 and the second from around residues 280-295.