Physical and kinetic properties of a pyridoxal reductase purified from bakers' yeast.

Pyridoxine dehydrogenase (1.1.1.65) (pyridoxal reductase), purified to homogeneity from baker's yeast, is a monomer of Mr approximately 33,000. It catalyzes the reversible oxidation of pyridoxine by NADP to yield pyridoxal and NADPH; equilibrium lies far in the direction of pyridoxine formation (Keq approximately 1.4 X 10(11) l/mol at 25 degrees C). Reduction of pyridoxal occurs most rapidly at pH 6.0-7.0; oxidation of pyridoxine is optimal at pH 8.6. NAD and NADH do not replace NADP and NADPH as substrates; pyridoxine, pyridoxal and pyridoxal 5'-phosphate are the only naturally occurring cosubstrates found. Several other aromatic aldehydes also are reduced, but substrate specificity and other properties of the enzyme distinguish it clearly from other alcohol dehydrogenases or aldehyde reductases. Between pH 6.3 and 7.1 (the intracellular pH of yeast), V/Km with pyridoxal and NADPH as substrates is greater than 600 times that observed with pyridoxine and NADPH as substrates is greater than 600 times that observed with pyridoxine and NADP as substrates. These and other considerations strongly indicate that the dehydrogenase functions in vivo to reduce pyridoxal to pyridoxine, which is the preferred substrate for pyridoxal (pyridoxine) kinase in yeast.

Purification and properties of pyridoxal-5'-phosphate-dependent histidine decarboxylases from Klebsiella planticola and Enterobacter aerogenes.

Histidine decarboxylases from Klebsiella planticola and Enterobacter aerogenes were purified to homogeneity and compared with the histidine decarboxylase from Morganella morganii. All three enzymes required pyridoxal 5'-phosphate as a coenzyme, showed optimal activity at pH 6.5, decarboxylated only histidine among the amino acids derived from protein, and were tetramers or dimers of identical subunits. Amino-terminal sequences of the three enzymes showed up to 81% homology through residue 33, but the enzymes differed sufficiently in amino acid composition and sequence so that no cross-reaction occurred between the K. planticola or E. aerogenes enzymes and antibodies to the decarboxylase from M. morganii. All three enzymes were inhibited by carbonyl reagents; by amino-, carboxyl-, and some methyl-substituted histidines; and by alpha-fluoromethylhistidine. These decarboxylases, all from gram-negative organisms, differed greatly in subunit structure, biogenesis, and other properties from the pyruvoyl-dependent histidine decarboxylases from gram-positive organisms described previously.

Purification and properties of a pyridoxal 5'-phosphate-dependent histidine decarboxylase from Morganella morganii AM-15.

A pyridoxal 5'-phosphate-dependent histidine decarboxylase from Morganella morganii AM-15 was purified to homogeneity. The enzyme is a tetramer (Mr 170,000) of identical subunits and binds 4 pyridoxal-P/tetramer; it is resolved by dialysis against cysteine at pH 6.8. Between pH 6.2 and 8.8, the holoenzyme shows pH-independent absorbance maxima at 333 and 416 nm. Vmax/Km is highest at pH 6.5; this optimum reflects chiefly increased Km values for histidine at lower or higher pH values, whereas Vmax is highest at pH 5.0 and decreases only moderately between pH 5.0 and 8.0. The enzyme also decarboxylates beta-(2-pyridyl)alanine and N tau-methylhistidine (but not N pi-methylhistidine); arginine, lysine, and ornithine are neither substrates nor inhibitors. The hydrazine analogue of histidine, 2-hydrazino-3-(4-imidazolyl)propionic acid, is a very potent competitive inhibitor; other carbonyl reagents and a variety of carboxyl- or amino-substituted histidines also inhibit competitively. alpha-Fluoromethylhistidine is a potent irreversible inhibitor of the enzyme; alpha-methylhistidine is a competitive inhibitor/substrate that is decarboxylated slowly and undergoes a slow decarboxylation-dependent transamination that converts the holoenzyme to pyridoxamine-P and apoenzyme. Dithiothreitol and other simple thiols are mixed-type inhibitors that interact with pyridoxal-P at the active site to form complexes (lambda max congruent to 340 nm), presumably the corresponding thioalkylamines, without resolving the holoenzyme. This histidine decarboxylase (Vmax = 72 mumol X min-1 X mg-1) is much more active than "homogeneous" preparations of mammalian pyridoxal-P-dependent histidine decarboxylase (Vmax congruent to 1.0) and is about equal in activity to the pyruvoyl-dependent histidine decarboxylases from Gram-positive bacteria.

Purification and properties of ornithine decarboxylase from Lactobacillus sp. 30a.

Inducible ornithine decarboxylase from Lactobacillus sp. 30a has been purified to homogeneity as judged by ultracentrifugation and gel electrophoresis. Unlike histidine decarboxylase from the same species (a pyruvoyl enzyme), ornithine decarboxylase is a pyridoxal phosphate enzyme. The purified enzyme is specific for L-pornithine (Km 1.7 mM; specific activity, 150 to 200 mumol min-1 mg-1 at 37 degrees C) and is inhibited by various homologous omega-amino acids, amines, and polyamines. The native enzyme has an isoelectric point of 4.55 and a molecular weight of 1.04 X 10(6). At pH 7.3 and above, it dissociates reversibly to a species of Mr = 184,000, and on gel electrophoresis in the presence of sodium dodecyl sulfate shows a single band of Mr = 85,000. We ascribe these species to the dodecamer, dimer, and monomer, respectively, of a single peptide subunit; electron micrographs show a hexagonal array of apparently dimeric subunits in the native enzyme. Highest enzymatic activity is present in the dodecamer. The holoenzyme is resolved by dialysis against cysteine; spectrophotoemetric titration of the apoenzyme with pyridoxal 5'-phosphate indicates the presence of 1 coenzyme-binding site/monomeric subunit.

The behavior of muscle phosphorylase as a reservoir for vitamin B6 in the rat.

Current belief that vitamin B6 deficiency causes depletion of muscle phosphorylase in animals appears to be erroneous. We present evidence that vitamin B6 deficiency is ineffective in reducing total phosphorylase in gasttocnemius muscle of young rats over a period of at least 8 weeks. Rats that had accumulated high levels of muscle phosphorylase while ingesting diets containing normal or excess amounts of the vitamin retained their phosphorylase after transfer to a vitamin B6 deficient diet. Prolonged deficiency did ultimately lead to enzyme depletion but this was after anorexia had developed and weight loss had occurred. When rats were partially starved for 1 to 4 days (fed 10% of normal energy intake) they lost muscle phosphorylase while retaining alanine and aspartate aminotransferases. When totally starved, the rats lost more phosphorylase than during partial starvation, but completely retained alanine aminotransferase, and lost some aspartate aminotrasferase. We conclude that the behavior of muscle phosphorylase is consistent with the Krebs-Fischer proposal that it acts as a reservoir for vitamin B6 and that starvation, but not vitamin B6 deficiency per se, causes depletion of muscle phosphorylase. It appears that phosphorylase may function as an adjunct ot adipose tissue necessary for the animal to efficiently meet the exigencies of starvation.

Increased muscle phosphorylase in rats fed high levels of vitamin B6.

The present study was undertaken to test the hypothesis that muscle phosphorylase may function as a repository for vitamin B6 in the animal. Since a repository would be expected to accumulate surplus material, one would predict that phosphorylase, which contains stoichio-metric amounts or pyridoxal phosphate, would increase in muscle of animals surfeited with the vitamin. Rats were fed a vitamin B6-free diet supplemented with pyridoxine providing levels 10, 1.0 and 0.1 of those recommended by the National Research Council (NRC). At the high intake level, muscle phosphorylase and total muscle vitamin B6 increased steadily and in almost constant ratio for at least 6 weeks, whereas both alanine and aspartate transaminase increased initially, but reached a plateau within 2 weeks. At the intermediate level of pyridoxine intake, muscle phosphorylase also increased, but less rapidly than in rats fed the higher level. When vitamin B6 intake was restricted to 10% of the NRC-recommended level, no increase in phosphorylase concentration occurred during a period of 10 weeks. These results support the hypothesis that muscle phosphorylase acts as a reservoir for vitamin B6 in the animal and provide experimental evidence that muscle enzyme content expands as vitamin is accumulated during high dietary intake.

Growth characteristics of a pseudomonad which utilizes pyridoxine or pyridoxamine as a carbon source.

Cultural characteristics of Pseudomonas MA-1, which has been extensively used as a source of enzymes that degrade vitamin B(6), confirm its classification as a pseudomonad. The organism requires biotin for growth; any one of several amino acids is also required for growth when pyridoxine (but not when glucose) serves as the energy source. Growth of Pseudomonas MA-1 is inhibited by one or more unidentified substances formed when pyridoxine is autoclaved with the medium. Modified cultural conditions are described which permit the large-scale growth of the organism with pyridoxine as carbon source.

Salmonella typhimurium mutants with alternate requirements for vitamin B 6 or isoleucine.

Several mutants of Salmonella typhimurium LT-2, isolated as auxotrophs for vitamin B(6), grew without the added vitamin when supplied with either isoleucine, alpha-ketobutyrate, or alpha-keto-beta-methylvalerate, but not with threonine or with other alpha-keto acids. When grown on minimal medium supplemented with isoleucine, these mutants synthesized vitamin B(6) in amounts comparable to wild-type cells; they thus appeared to contain a modified l-threonine dehydratase and to belong to genotype ilvA (threonine dehydratase) instead of pdx (pyridoxine). Direct assays confirmed this hypothesis. Wild-type cells (toluene-treated) showed approximately the same threonine dehydratase activity whether grown in the presence or absence of added pyridoxal-P; mutant cells approached the activity of wild-type cells only when they were grown with added vitamin B(6) and were assayed in the presence of pyridoxal-P. In cell-free extracts, the threonine dehydratase from mutant cells was cold labile and more labile to oxidative inactivation than the wild-type enzyme; furthermore, activation of the mutant apoenzyme required a 10- to 20-fold higher concentration of pyridoxal-P than was required for the wild-type apoenzyme. These results show that cultures which appear auxotrophic for a given vitamin may synthesize that vitamin in normal amounts, the exogenous requirement arising from impaired binding of the vitamin-derived coenzyme to a genetically altered apoenzyme dependent on that coenzyme. Inadequate nutritional data to support the genetic findings can lead to erroneous genotype classification for such mutants.