Shoichi Shimizu

Properties and catalytic activities of vitamin B6 N-oxide☆

Abstract To obtain further information on the role of the pyridine-nitrogen of vitamin B 6 in nonenzymatic and enzymatic systems, the N-oxides of pyridoxal, pyridoxamine, and their 5′-phosphates were synthesized and their catalytic actions studied. In the nonenzymatic α,β-elimination of tryptophan and serine, pyridoxal N-oxide served as a catalyst, though somewhat less efficiently than pyridoxal. Transamination reaction between pyridoxal N-oxide and glutamate occurred only slightly. On the other hand, the reaction between pyridoxamine N-oxide and α-ketoglutarate proceeded readily, but at a slower rate than when pyridoxamine was used. The lower activities of the N-oxides could be ascribed to the decrease in electronegativity of the pyridine-nitrogen. Pyridoxal N-oxide 5′-phosphate served as the coenzyme for E. coli tryptophanase. Although its K m value was nearly equal to that of pyridoxal phosphate, the maximum velocity was only 65% of that of the native coenzyme. In a mammalian glutamic-oxaloacetic transaminase (GOT) system, the activity of the coenzyme analogue-bound apoenzyme varied with the reaction time even after a 10-min preincubation for the reconstitution. In this case, a plot of the reaction velocity vs. coenzyme concentration gave a sigmoidal curve, suggesting a complicated interaction between the coenzyme analogue and the apoenzyme.

A cobalt-free corrinoid compound in Streptomyces olivaceus.

Abstract An unknown orange-red pigment was obtained from the cells of Streptomyces olivaceus 605 grown on a cobalt-free medium. This compound showed similar behaviors to those of the cobalt-free corrinoids of Chromatium isolated by Toohey in UV, CD and fluorescence spectra. However it exhibited a more acidic character in paper electrophoresis. Although an attempted insertion of cobalt atom has been unsuccessful, available evidence indicates that this pigment is the first cobalt-free corrinoid found in nonphotosynthetic bacteria, having carboxylic acid side-chains on its corrin nucleus.

Formation of vitamin B12s by anaerobic photolysis of cobalt-alkylcobalamins

i.e., the irradiated solution as well as authentic methionine gave a spot at RF 0.48 in n-butanol-acetic acid-water (12:4:7, by vol.), detected by spraying with the ninhydrin reagent, p-Benzoquinone, a well-known radical acceptor, is also considered to trap the methyl radical. Thus it is concluded that in the presence of compounds which are readily attacked by radicals, cobalt-methylcobalamin is easily photolyzed. The effect of O 3 would be partially explained by the same consideration. In the case of the 5,6dimethylbenzimidazolyl cobamide-coenzyme and cobait-5-deoxy-2,3-isopropylideneuridinylcobalamin, it seems important that C-8 of the 5-deoxyadenosyl ligand in the former and C-6 of the 5-deoxy-2,3-isopropylideneuridinyl ligand in the latter are open to radical attack.

Comparison of reactivity between D-propanediol and L-propanediol in intramolecular oxidation-reduction catalyzed by dioldehydrase requiring cobamide coenzyme.

Abstract The substrate stereospecificity of a dioldehydrase requiring DBC-coenzyme was investigated with d (−)- and l (+)-propanediols as the substrates. Both isomers could be converted to propionaldehyde, but the d -isomer was 1.5–2 times more reactive than the l -form. An inhibition was observed if a high concentration of the l -form or the dl -form was used; moreover, the reactivity of the d -form was depressed by the simultaneous addition of the l -form.

Reduction of cobalt atom of corrinoid compounds by alkali treatment

Abstract 1. 1. When cyanocobalamin or 8-aminocobyrinic acid- c -lactam were heated in 30% NaOH at 120° for 1 h, a green substance was formed whose spectrum was similar to that of vitamin B 12s , and this substance readily reacted with emtyyl iodide to yield cobalt-methyl-8-aminocobyrinic acid- c -lactam as the main product. 2. 2. Cyanocobalamin heated anaerobically in 0.4% NaOH at 100° for 10 min gave a yellow-brown substance whose spectrum ws similar to that of vitamine B 12r , and this substance reacted with methyl iodide of n -butyl bromide to yield cobalt-methyl or cobalt- n -butyl derivatives of dehydrocobalamin, respectively, and their mono- to tricarboxylic acid derivatives. 3. 3. On being heated anaerobically in 0.4% NaOH, cyanocobalamin and aquo(or hydroxo)-cobalamin rapidlyturned yellow-brow, while cyanodehydrocobalamin and aquo(or hydroxo)-dehydrocobalamin changed only verly slowly. From these facts it seems probable that the occurrence of the yellow-brown state with the behaviour of vitamin B 12r may be due to the reduction of the cobalt atom accompanied by dehydrogenation on the B ring of the corrin nucleus. 4. 4. The formation of the green substance from 8-aminocobyrinic acid- c -lactam on vigorous alkali treatment is, however, interpreted by assuming another reaction mechanism causing further reduction of the cobalt atom.

Metabolism of Glutamate in Purple Nonsulfur Bacteria Participation of Vitamin B12

Purple nonsulfur bacteria, Rhodospirillum rubrum and Rhodopseudomonas spheroides were found to possess coenzyme B12-dependent glutamate mutase activity. Cell-free extracts of these bacteria grown on Co2+-containing media catalyzed the conversion of glutamate to β-methylaspartate and further to mesaconate. The activity of the cell-free extracts of these organisms cultivated on Co2+-deficient media was markedly lower than that of the normal cells. Addition of coenzyme B12 to the former reaction mixture enhanced the mesaconate formation via β-methylaspartate. These results indicate the involvement of coenzyme Independent glutamate mutase of these bacteria in the dissimilation of glutamate to acetyl-CoA and pyruvate through the following pathway.glutamate→β→methylaspartate→mesaconate→citramalate→→acetyl-CoA, pyruvate On the other hand, a greater part of glutamate was converted to α-hydroxyglutarate and succinate with the cell-free extracts of these photosynthetic bacteria. This fact, taking account of the pre...

Comparison of coenzymatic activities of 2-nor-2-hydroxymethyl pyridoxal 5′-phosphate for mitochondrial and cytoplasmic aspartate aminotransferases

Abstract 2-Nor-2-hydroxymethyl pyridoxal 5′-phosphate served as a somewhat superior coenzyme to pyridoxal 5′-phosphate for cytoplasmic and mitochondrial aspartate aminotransferases in the usual assay system containing Tris-HCl buffer. In the presence of phosphate anion, however, the affinities of the analog for both enzymes and the V values of the reactions mediated by the cytoplasmic and mitochondrial aspartate aminotransferases reconstituted with the analog were more markedly decreased than in the cases of the native coenzyme. Various Vitamin B 6 enzymes tested hitherto were classified into at least three groups according to their attitudes toward the coenzyme analog.

Studies on the Corrinoids and Porphyrins in Streptomycetes :Part III. Influence of Cultural Conditions on the Forms of Corrinoids Produced by Streptomyces olivaceus

When Streptomyces olivaceus 605 was cultivated in a medium containing glycerol as sole carbon source, it was found to produce three kinds of corrinoids, i.e. 5′-deoxyadenosylcobalamin (DBCC), 5′-deoxyadenosylcobinamide (CC), and methylcobalamin (Me-B12). The amount of these corrinoids changed markedly according to the cultural conditions. When cobalt ion was added at the optimal level to form large amounts of corrinoids, CC was the most predominant form in the glycerol medium. On the other hand, DBCC was the main corrinoid formed in the glucose-lactose medium. The forms of corrinoids varied also during the cultivation period. The ratio of the amount of Me-B12 to the total corrinoids was significantly high in the early stage of the growth, while DBCC was predominant after the later exponential phase. Under the cobalt-deficient conditions in both the glycerol medium and the glucose-lactose medium, the amount of CC was drastically decreased.

Studies on the Corrinoids and Porphyrins in Streptomycetes: Part IV. Confirmation of a Cobalamin-dependent Methionine Synthesizing System in Streptomyces olivaceusPart V Effect of Additions on Porphyrin Excretion and δ-Aminolevulinic Acid Dehydratase in Streptomyces olivaceus

This paper deals with the confirmation of the existence of a cobalamin-dependent terminal step in the methionine synthesis, that is, the methyl transfer from N5-CH3-H4-folate to homocysteine to form methionine, in the cell-free extracts of Streptomyces olivaceus 605.This transmethylation reaction required a reducing system and S-adenosylmethionine (SAM) as cofactors.Methionine formation from serine was observed in the cell-free system and thus this reaction is considered to participate in a series of one-carbon metabolism from serine.

Studies on the Corrinoids and Porphyrins in Streptomycetes: Part I. Characterization of 5, 6-Dimethylbenzimidazolylcobamide Coenzyme Formed in the Cells of Streptomyces olivaceusPart II. Factors Influencing the Accumulation of Coproporphyrin III in the Culture Filtrate of Streptomyces olivaceus 605

A corrinoid, formed predominantly in the cells of Streptomyces olivaceus 605 grown aerobically on a glucose-lactose medium, was identical with 5,6-dimethylbenzimidazolyl-cobamide coenzyme (DBC coenzyme) by paper chromatography, paper ionophoresis, absorption spectrophotometry, assay of the coenzyme activity in diol dehydratase system and other tests.Immediately after the maximal growth, the corrinoid was excreted into the cultural filtrate and converted to various unidentified forms of corrinoids.In a medium containing glycerol as a carbon source, Streptomyces olivaceus 605 accumulated under mild aeration a large amount of porphyrin in the culture filtrate. Identity of methyl ester of the porphrin with coproporphyrin III methyl ester was confirmed by UV-, IR- and NMR- spectroscopy. Under the cultural conditions favarable for production of corrinoid, the accumulation of coproporphyrin III markedly decreased.