Active site structure and stereospecificity of Escherichia coli pyridoxine-5'-phosphate oxidase.

Pyridoxine-5'-phosphate oxidase catalyzes the oxidation of either the C4' alcohol group or amino group of the two substrates pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate to an aldehyde, forming pyridoxal 5'-phosphate. A hydrogen atom is removed from C4' during the oxidation and a pair of electrons is transferred to tightly bound FMN. A new crystal form of the enzyme in complex with pyridoxal 5'-phosphate shows that the N-terminal segment of the protein folds over the active site to sequester the ligand from solvent during the catalytic cycle. Using (4'R)-[(3)H]PMP as substrate, nearly 100 % of the radiolabel appears in water after oxidation to pyridoxal 5'-phosphate. Thus, the enzyme is specific for removal of the proR hydrogen atom from the prochiral C4' carbon atom of pyridoxamine 5'-phosphate. Site mutants were made of all residues at the active site that interact with the oxygen atom or amine group on C4' of the substrates. Other residues that make interactions with the phosphate moiety of the substrate were mutated. The mutants showed a decrease in affinity, but exhibited considerable catalytic activity, showing that these residues are important for binding, but play a lesser role in catalysis. The exception is Arg197, which is important for both binding and catalysis. The R197 M mutant enzyme catalyzed removal of the proS hydrogen atom from (4'R)-[(3)H]PMP, showing that the guanidinium side-chain plays an important role in determining stereospecificity. The crystal structure and the stereospecificity studies suggests that the pair of electrons on C4' of the substrate are transferred to FMN as a hydride ion.     

Pyridoxal phosphate inhibits the DNA-binding activity of the ecdysteroid receptor

The effect of pyridoxal 5'-phosphate on the binding of the ecdysteroid receptor from a nuclear extract of Drosophila melanogaster to DNA-cellulose was studied. The binding of hormone-receptor complexes to DNA-cellulose was completely blocked after a 30-min incubation with 3 mM pyridoxal 5'-phosphate at 0-4 degree C. The effect was specific for pyridoxal 5'-phosphate since related compounds (pyridoxal, pyridoxamine 5'-phosphate and pyridoxamine) were not effective or gave only 17% inhibition (pyridoxal). Under standard conditions, none of the compounds tested exerted a significant effect on the stability of [3H](20R,22R)-2 beta,3 beta, 14 alpha,20,22-pentahydroxy-5 beta-cholest-7-en-6-one ([3H]ponasterone A)-receptor complexes. The loss of DNA-binding activity caused by pyridoxal 5'-phosphate is accompanied by changes in the molecular properties of [3H]ponasterone-A-receptor complexes. A shift of [3H]ponasterone-A binding was observed from the 8.0-8.5 S to the 4.5-5.0 S region, when [3H]ponasterone-A-receptor complexes were exposed to pyridoxal 5'-phosphate during sucrose-gradient centrifugation. The inhibition of DNA-cellulose binding by pyridoxal 5'-phosphate can be reversed. Probably, pyridoxal 5'-phosphate forms a Schiff base with a critical lysine group of the ecdysteroid receptor, presumably at its DNA-binding site. The hormone-receptor complexes obtained after removal of pyridoxal 5'-phosphate had the same affinity for DNA-cellulose as 'native' complexes. DNA-cellulose-bound [3H]ponasterone-A complexes were efficiently eluted from DNA-cellulose with pyridoxal 5'-phosphate in 0.1 M KCl resulting in a 104-fold purification of the ecdysteroid receptor. The results reflect possible structural similarities between ecdysteroid and vertebrate steroid receptors.     

Pyridoxine-derived B6 vitamers and pyridoxal 5'-phosphate-binding proteins in cytosolic and nuclear fractions of HTC cells

The nuclear fraction of rat hepatoma-derived HTC cells contained approximately 8% of the total cellular pyridoxal 5'-phosphate. HTC cells were able to metabolize [3H]pyridoxine to coenzymatically active pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate. As HTC cells did not have any demonstrable pyridoxine-5'-phosphate oxidase activity, the conversion of pyridoxine to pyridoxal 5'-phosphate must have taken place by a nonconventional route. The ratio of pyridoxal 5'-phosphate to pyridoxamine 5'-phosphate in the nonnuclear fraction of HTC cells was approximately 1:1, whereas in the nuclear fraction it was approximately 17:1, indicating that there was selective acquisition of pyridoxal 5'-phosphate by the nucleus. With the aid of a monoclonal antibody specific for the 5'-phosphopyridoxyl group, it was shown that there was one major pyridoxal 5'-phosphate-binding protein in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-resolved nucleoplasmic extract of HTC cells. This finding was confirmed by radioautography of an SDS-PAGE-resolved nucleoplasmic extract obtained from cells grown in a medium containing [3H]pyridoxine. Isoelectric focusing followed by SDS-PAGE also indicated the presence of one major pyridoxal 5'-phosphate-binding protein in the nucleoplasmic extract of HTC cells having a relatively high isoelectric point (approximately 7). Data were obtained indicating that the protein might exist in a higher molecular weight form, probably a dimer. Currently, these findings constitute virtually all of the available information on vitamin B6 and the cell nucleus.     

Stereospecific labilization of the C-4' pro-S hydrogen of pyridoxamine 5'-phosphate in aspartate aminotransferase

In the course of a half-reaction of enzymic transamination, the aldimine adduct formed between the coenzyme pyridoxal 5'-phosphate and the amino acid substrate tautomerizes to the ketimine intermediate which is then hydrolyzed to the oxo acid product and the pyridoxamine 5'-phosphate form of the enzyme. In the reverse half-reaction the tautomerization is initiated by the removal of a proton from the pro-S position at C-4' of the PMP moiety of the ketimine intermediate. The present study investigates the question whether the pro-S hydrogen at C-4' of PMP is labilized by its active site environment independently of the formation of the ketimine intermediate, i.e. in the absence of substrate. Reconstitution of apoaspartate aminotransferase (mitochondrial isoenzyme from chicken) with [4'-3H] PMP results indeed in a stereospecific exchange of pro-S 3H with solvent water. The exchange follows first order kinetics (t 1/2 = 23 min at pH 7.5 and 25 degrees C). Unbound PMP showed no measurable exchange. Rigorous control experiments excluded the possibility that the observed exchange was due to a transamination reaction of the enzyme with contaminating oxo acid substrates. The newly observed stereospecific exchange reaction allows to investigate the acid/base properties of C-4' and the modulating effects of its active site environment independently of the preceding and following steps of enzymic transamination.     

Kinetic studies of the pyridoxal kinase from pig liver: slow-binding inhibition by adenosine tetraphosphopyridoxal

Pyridoxal kinase from pig liver has been purified 10,000-fold to apparent homogeneity. The enzyme is a dimer of subunits of Mr 32,000. The enzyme is strongly inhibited by the product pyridoxal 5'-phosphate. Liver pyridoxamine phosphate oxidase, another enzyme involved in the biosynthesis of pyridoxal 5'-phosphate, is also strongly inhibited by this compound [Wada, H., & Snell, E. E. (1961) J. Biol. Chem. 236, 2089-2095]. Thus, the biosynthesis of pyridoxal 5'-phosphate in the liver might be regulated by the product inhibition of both pyridoxamine phosphate oxidase and pyridoxal kinase. Kinetic studies revealed that the catalytic reaction of liver pyridoxal kinase follows an ordered mechanism in which pyridoxal and ATP bind to the enzyme and ADP and pyridoxal 5'-phosphate are released from the enzyme, in this order. Adenosine tetraphosphopyridoxal was found to be a slow-binding inhibitor of pyridoxal kinase. Pre-steady-state kinetics of the inhibition revealed that the inhibitor and the enzyme form an initial weak complex prior to the formation of a tighter and slowly reversing complex. The overall inhibition constant was 2.4 microM. ATP markedly protects the enzyme against time-dependent inhibition by the inhibitor, whereas another substrate pyridoxal affords no protection. By contrast, adenosine triphosphopyridoxal is not a slow-binding inhibitor of this enzyme.     

Mechanism of reactions catalyzed by selenocysteine beta-lyase

The reaction mechanism of selenocystine beta-lyase has been studied and it was found that elemental selenium is released enzymatically from selenocysteine, and reduced to H2Se nonenzymatically with dithiothreitol or some other reductants that are added to prepare selenocysteine from selenocystine in the anaerobic reaction system. 1H and 13C NMR spectra of L-alanine formed in 2H2O have shown that an equimolar amount of [beta-2H1]- and [beta-2H2]alanines are produced. The deuterium isotope effect at the alpha position was observed; kH/kD = 2.4. These results indicated that the alpha hydrogen of selenocysteine was removed by a base at the active site, and was incorporated into the alpha position of alanine, a product, without exchange of a solvent deuterium. When the enzyme was incubated with L-selenocysteine in the absence of added pyridoxal 5'-phosphate, the activity decreased with prolonged incubation time. However, the activity was recovered by addition of 5'-phosphate. The spectrophotometric study showed that the inactivated enzyme was the apo form. The apoenzyme was activated by a combination of pyridoxamine 5'-phosphate and various alpha-keto acids such as alpha-ketoglutarate and pyruvate. Thus, the enzyme is inactivated through transamination between selenocysteine and the bound pyridoxal 5'-phosphate to produce pyridoxamine 5'-phosphate and a keto acid derived from selenocysteine. The pyridoxal enzyme, an active form, is regenerated by addition of alpha-keto acids. This regulatory mechanism is analogous to those of aspartate beta-decarboxylase [EC 4.1.1.12], arginine racemase [EC 5.1.1.9], and kynureninase [EC 3.7.1.3] [K. Soda and K. Tanizawa (1979) Adv. Enzymol. 49, 1].     

An enzymatic method for the synthesis of [14C]pyridoxal 5'-phosphate from [14C]pyridoxine

A new enzymatic method for the synthesis of [14C]pyridoxal 5'-phosphate is presented. [14C]Pyridoxal 5'-phosphate was synthesized from [14C]pyridoxine through the successive actions of pyridoxal kinase and pyridoxamine 5'-phosphate oxidase in a reaction mixture containing ATP, [14C]pyridoxine, and both enzymes. [14C]Pyridoxal 5'-phosphate was isolated by omega-aminohexyl-Sepharose 6B column chromatography. The overall yield of the product was more than 60%, starting from 550 nmol of [14C]pyridoxine. The radiochemical purity of the products, as determined by thin-layer and ion-exchange chromatography, was greater than 98%.     

Mechanism of inactivation of gamma-aminobutyric acid aminotransferase by (S,E)-4-amino-5-fluoropent-2-enoic acid

Evidence for an enamine mechanism of inactivation of pig brain gamma-aminobutyric acid (GABA) aminotransferase by (S,E)-4-amino-5-fluoropent-2-enoic acid is presented. apo-GABA aminotransferase reconstituted with [3H]pyridoxal 5'-phosphate is inactivated by (S,E)-4-amino-5-fluoropent-2-enoic acid and the pH is raised to 12. All of the radioactivity is released from the enzyme as an adduct of the cofactor; no [3H]pyridoxamine 5'-phosphate is generated.     

The stereospecific labilization of the C-4' pro-S hydrogen of pyridoxamine 5'-phosphate is abolished in (Lys258----Ala) aspartate aminotransferase

Reconstitution of wild-type apoaspartate aminotransferase from Escherichia coli with [4'-3H]pyridoxamine 5'-phosphate results in stereospecific release of the pro-S C-4' 3H to the solvent. The reaction follows first-order kinetics (t1/2 = 15 min at pH 7.5 and 25 degrees C), its rate constant being similar to that found previously with mitochondrial aspartate aminotransferase from chicken (Tobler, H.P., Christen, P., and Gehring, H. (1986) J. Biol. Chem. 261, 7105-7108). Substituting the active site residue Lys258 by alanine via site-directed mutagenesis yields a catalytically inactive enzyme (Malcolm, B. A., and Kirsch, J. F. (1985) Biochem. Biophys. Res. Commun. 132, 915-921). This mutant enzyme fails to release any measurable 3H from bound [4'-3H]pyridoxamine 5'-phosphate. The data are consistent with earlier proposals that Lys258 is indispensable for the ketimine/aldimine tautomerization, and corroborate the previous conclusion that 3H exchange from enzyme-bound pyridoxamine 5'-phosphate mechanistically corresponds to the deprotonation at C-4' of the ketimine intermediate during the transamination reaction.     

Identification of coenzyme aldimine proton in 1H NMR spectra of pyridoxal 5'-phosphate dependent enzymes: aspartate aminotransferase isoenzymes

The pyridoxal form of the alpha subform of cytosolic aspartate aminotransferase (EC 2.6.1.1) is fully active and binds pyridoxal 5'-phosphate via an aldimine formation with Lys-258 whereas the gamma subform is virtually inactive and lacks the aldimine linkage. Comparison of 1H NMR spectra between the alpha and gamma subforms suggested that peak 1 of the alpha subform at 8.89 ppm contains a resonance assignable to the internal aldimine 4'-H. Reaction with a reagent that cleaves or modifies the internal aldimine bond [(amino-oxy)acetate, L-cysteinesulfinate, NH2OH, NaBH4, or NaCNBH3] caused the disappearance of a resonance line at 8.89 ppm that possessed a broad line width and corresponded in intensity to a single proton. These reagents were also used successfully for the identification of the aldimine 4'-H resonance in the mitochondrial isoenzyme. In contrast to the cytosolic isoenzyme whose resonance for the 4'-H did not show any detectable change in chemical shift with pH, the corresponding resonance in the mitochondrial isoenzyme exhibited pH-dependent chemical shift change (8.84 ppm at pH 5 and 8.67 ppm at pH 8) with a pK value of 6.3, reflecting the interisozymic difference in the microenvironment provided for the internal aldimine. Validity of the signal assignment was further shown by the two findings: the resonance assigned to the 4'-H emerged upon conversion of the pyridoxamine into the pyridoxal form, and the resonance appeared upon reconstitution of the apoenzyme with [4'-1H]pyridoxal phosphate but not with [4'-2H]pyridoxal phosphate.     

EFFECT OF HYPERPHENYLALANINEMIA ON VITAMIN B6, METABOLISM IN DEVELOPING RAT BRAIN

Abstract— The turnover of the different forms of B₆ vitamers in the brains of normal and hyperphenylalaninemic preweanling rats was compared after administration of a load of [¹⁴C]pyridoxol. Metabolic transformations occurred in the following sequence: oxidation of pyridoxol to pyridoxal, which was in turn phosphorylated to the 5'-phosphate ester. No significant amount of pyridoxamine was formed during the 8-h experimental period. Pyridoxamine 5'-phosphate was derived from pyridoxal 5'-phosphate. The specific radioactivity of pyridoxal phosphate in the hyperphenylalaninemic brain was significantly lower and increased at a slower rate than in control brains. This difference could not be accounted for by either a deficient supply or inhibited activity of the enzyme, pyridoxal kinase. The synthesis of pyridoxamine 5'-phosphate in the experimental animals also lagged behind the controls. Decreased activity of enzymes dependent on pyridoxal phosphate as cofactor would explain the slower turnover of this B₆-coenzyme.     

Metabolism of pyridoxine in the liver of vitamin B-6-deficient rats.

The metabolism of [6-3H]pyridoxine - HCl was investigated in the liver of vitamin B-6-deficient rats. Rats were made vitamin B-6 deficient by feeding ad libitum for 42 days a diet lacking pyridoxine but otherwise optimal. Animals were each injected intraperitoneally with 33 muCi of [6-3H] pyridoxine - HCl and killed at different time intervals afterwards up to 7 days. Radioactively labeled hepatic B-6 compounds were extracted with acid and chromatographically separated on Dowex-X8 (H+) columns and the percent radioactivity for each vitamin compound was then calculated. Maximal uptake in control and deficient animals was observed 30 and 60 min, respectively, after administration of label. Radioactivity was not retained by the control animals but decreased steadily in a linear fashion after 30 min, reaching a low level after 3 h. On the other hand, vitamin deficient animals accumulated almost twice as much radioactivity in their liver as the controls and retained it through 7 days. In vitamin B-6 deficient animals 93% of the injected radioactivity was metabolized within 2 min at which time pyridoxine 5'-P and pyridoxal 5'-P reached 36 and 44% levels, respectively. Pyridoxine 5'-P dropped to minimal values (3%) within 15 min and remained unchanged for 7 days while pyridoxal 5'-P reached a peak (79%) level at 15 min and then began to drop linearly reaching a plateau (29%) at 5 days. Further, as the level of pyridoxal 5-P was falling, pyridoxamine 5'-P was linearly synthesized reaching a platuau low level (3%). The specific activity level of pyridoxal kinase decreased 3.2 times and that of pyridoxine 5'-phosphate oxidase increased 1.5 times in the state of deficiency. The results presented show that metabolism of [3H]pyridoxine in deficiency is characterized by (a) a delayed, two-fold increase in label uptake as well as an extended label retention period, (b) a rapid pyridoxal 5'-P synthesis, and (c) a continuous synthesis (and accumulation) of pyridoxamine 5'-P which is not utilized or further metabolized.     

Tyrosine 265 of alanine racemase serves as a base abstracting alpha-hydrogen from L-alanine: the counterpart residue to lysine 39 specific to D-alanine.

Alanine racemase of Bacillus stearothermophilus has been proposed to catalyze alanine racemization by means of two catalytic bases: lysine 39 (K39) abstracting specifically the alpha-hydrogen of D-alanine and tyrosine 265 (Y265) playing the corresponding role for the antipode L-alanine. The role of K39 as indicated has already been verified [Watanabe, A., Kurokawa, Y., Yoshimura, T., Kurihara, T., Soda, K., and Esaki, N. (1999) J. Biol. Chem. 274, 4189-4194]. We here present evidence for the functioning of Y265 as the base catalyst specific to L-alanine. The Y265-->Ala mutant enzyme (Y265A), like Y265S and Y265F, was a poor catalyst for alanine racemization. However, Y265A and Y265S catalyzed transamination with D-alanine much more rapidly than the wild-type enzyme, and the bound coenzyme, pyridoxal 5'-phosphate (PLP), was converted to pyridoxamine 5'-phosphate (PMP). The rate of transamination catalyzed by Y265F was about 9% of that by the wild-type enzyme. However, Y265A, Y265S, and Y265F were similar in that L-alanine was inert as a substrate in transamination. The apo-form of the wild-type enzyme catalyzes the abstraction of tritium non-specifically from both (4'S)- and (4'R)-[4'-(3)H]PMP in the presence of pyruvate. In contrast, apo-Y265A abstracts tritium virtually from only the R-isomer. This indicates that the side-chain of Y265 abstracts the alpha-hydrogen of L-alanine and transfers it supra-facially to the pro-S position at C-4' of PMP. Y265 is the counterpart residue to K39 that transfers the alpha-hydrogen of D-alanine to the pro-R position of PMP.     

Structure and mechanism of Escherichia coli pyridoxine 5'-phosphate oxidase

Escherichia coli pyridoxine 5'-phosphate oxidase (PNPOx) catalyzes the oxidation of either pyridoxine 5'-phosphate (PNP) or pyridoxamine 5'-phosphate (PMP), forming pyridoxal 5'-phosphate (PLP). This reaction serves as the terminal step in the de novo biosynthesis of PLP in E. coli and as a part of the salvage pathway of this coenzyme in both E. coli and mammalian cells. Recent studies have shown that in addition to the active site, PNPOx contains a noncatalytic site that binds PLP tightly. The crystal structures of PNPOx with one and two molecules of PLP bound have been determined. In the active site, the PLP pyridine ring is stacked almost parallel against the re-face of the middle ring of flavin mononucleotide (FMN). A large protein conformational change occurs upon binding of PLP. When the protein is soaked with excess PLP an additional molecule of this cofactor is bound about 11 A from the active site. A possible tunnel exists between the two sites. Site mutants were made of all residues at the active site that make interactions with the substrate. Stereospecificity studies showed that the enzyme is specific for removal of the proR hydrogen atom from the prochiral C4' carbon of PMP. The crystal structure and the stereospecificity studies suggest that the pair of electrons on C4' of the substrate are transferred to FMN as a hydride ion.     

Effect of pyridoxal 5'-phosphate on Ca2+-stimulated adenosine triphosphatase activity in microsomes of rat submandibular gland in vitro

1. The Ca2+-ATPase activity in microsomes of rat submandibular gland was inhibited by pyridoxal 5'-phosphate in vitro. 2. The dissociation constant of the enzyme-pyridoxal 5'-phosphate complex was estimated to be 6.5 mM. 3. The inhibition of pyridoxal 5'-phosphate for both ATP and Ca2+ was competitive. 4. The order of inhibitory effectiveness of pyridoxal 5'-phosphate analogs was pyridoxal 5'-phosphate greater than pyridoxal HCl greater than pyridoxamine 5'-phosphate greater than pyridoxamine HCl. 5. The enzyme-pyridoxal 5'-phosphate complex was nonreducible with sodium borohydride.     

Activity of Pyridoxamine as a Substrate for Brain Pyridoxal Kinase

The substrate activity of pyridoxamine (PM) for brain pyridoxal (PL) kinase was examined in view of a recent report which indicated that PM was a poor substrate for this enzyme. Bovine brain PL kinase was shown by liquid chromatography to catalyze the phosphorylation of PM (Km = 65 microM). The identity of the reaction product, pyridoxamine 5'-phosphate, was confirmed by is ability to act as a substrate for liver pyridoxine (pyridoxamine) 5'-phosphate oxidase. The results, which indicate that PM is a good substrate for brain PL kinase, are consistent with the proposed role of intracellular phosphorylation in the uptake of vitamin B-6 brain tissue.     

Uptake and metabolism of N-(4'-pyridoxyl)amines by isolated rat liver cells.

Uptake and metabolism of [3H]pyridoxine and 3H-labeled N-(4'-pyridoxyl)amines by isolated rat liver cells were studied at physiological concentration (0.5 microM) of vitamin B6 by using both membrane filtration and centrifugation methods for removal of radiolabeled solutes after incubations with cells. It was found that the characteristics of import of N-(4'-pyridoxyl)amines into liver cells is similar to those of import of natural vitamin B6. Upon entry each 4'(N)-substituted pyridoxamine was converted to its 5'-phosphate and then oxidized to release pyridoxal 5'-phosphate and the original amine. Considerable size of the amine substituent is tolerated for transport and metabolism, but a charged function impedes entry. The amount of released pyridoxal 5'-phosphate (and therefore the amount of released original amine) is controlled partially by the size of the amine affixed to B6 and partially by the enzymatic steps involved. This system illustrates how biologically active amines can be piggybacked onto a vitamin that gains facilitated entry to cells that have the enzymatic means to release the free amine for subsequent effects within the cell.     

13C NMR spectroscopy of labeled pyridoxal 5'-phosphate. Model studies, D-serine dehydratase, and L-glutamate decarboxylase.

Pyridoxal 5'-phosphate labeled to the extent of 90% with 13C in the 4' (aldehyde) and 5' (methylene) positions has been synthesized. 13C NMR spectra of this material and of natural abundance pyridoxal 5'-phosphate are reported, as well as 13C NMR spectra of the Schiff base formed by reaction of pyridoxal 5'-phosphate with n-butylamine, the secondary amine formed by reduction of this Schiff base, the thiazolidine formed by reaction of pyridoxal 5'-phosphate with cysteine, the hexahydropyrimidine formed by reaction of pyridoxal 5'-phosphate with 1,3-diaminobutane, and pyridoxamine 5'-phosphate. The range of chemical shifts for carbon 4' in these compounds is more than 100 ppm, and thus this chemical shift is expected to be a sensitive indicator of structure in enzyme-bound pyridoxal 5'-phosphate. The chemical shift of carbon 5', on the other hand, is insensitive to these structure changes. 13C NMR spectra have been obtained at pH 7.8 and 9.4 for D-serine dehydratase (Mr = 46,000) containing natural abundance pyridoxal 5'-phosphate and containing 13C-enriched pyridoxal 5'-phosphate. The enriched material contains two new resonances not present in the natural abundance material, one at 167.7 ppm with a linewidth of approximately 24 Hz, attributed to carbon 4' of the Schiff base in the bound coenzyme, and one at 62.7 Hz with a linewidth of approximately 48 Hz attributed to carbon 5' of the bound Schiff base. A large number of resonances due to individual amino acids are assigned. The NMR spectrum changes only slightly when the pH is raised to 9.4. The widths of the two enriched coenzyme resonances indicate that the coenzyme is rather rigidly bound to the enzyme but probably has limited motional freedom relative to the protein. 13C NMR spectra have been obtained for L-glutamate decarboxylase containing natural abundance pyridoxal 5'-phosphate and 13C-enriched pyridoxal 5'-phosphate. Under conditions where the two enriched 13C resonances are clearly visible in D-serine dehydratase, no resonances are visible in enriched L-glutamate decarboxylase, presumably because the coenzyme is rigidly bound to the protein and the 300,000 molecular weight of this enzyme produces very short relaxation times for the bound coenzyme and thus very broad lines.     

Stereospecificity of sodium borohydride reduction of tyrosine decarboxylase from Streptococcus faecalis.

Sodium boro[3H]hydride reduction of tyrosine decarboxylase from Streptococcus faecalis followed by complete hydrolysis of the enzyme produces epsilon-[3H]pyridoxyllysine. Degradation of this material to [4'-3H]pyridoxamine and stereochemical analysis with apoaspartate aminotransferase shows that the re side at C-4' of the cofactor is exposed to solvent at pH 5.5 and 7.0. After binding of L-tyrosine at pH 5.5 or tyramine at pH 7.0 to the holoenzyme, sodium boro[3H]hydride reduction proceeds from the si face at C-4' of the substrate . cofactor complex. This indicates one of two conformational changes occurs upon binding of substrate; either rotation about the C-4 to C-4' bond in the cofactor or rotation about the axis through the C-5 and C-5' bond.     

Rv2607 from Mycobacterium tuberculosis is a pyridoxine 5'-phosphate oxidase with unusual substrate specificity

Despite intensive effort, the majority of the annotated Mycobacterium tuberculosis genome consists of genes encoding proteins of unknown or poorly understood function. For example, there are seven conserved hypothetical proteins annotated as homologs of pyridoxine 5'-phosphate oxidase (PNPOx), an enzyme that oxidizes pyridoxine 5'-phosphate (PNP) or pyridoxamine 5'-phosphate (PMP) to form pyridoxal 5'-phosphate (PLP). We have characterized the function of Rv2607 from Mycobacterium tuberculosis H37Rv and shown that it encodes a PNPOx that oxidizes PNP to PLP. The k(cat) and K(M) for this reaction were 0.01 s(-1) and 360 μM, respectively. Unlike many PNPOx enzymes, Rv2607 does not recognize PMP as a substrate.