Influence of isoleucine upon quinomycin biosynthesis by Streptomyces sp. 732

When dl-isoleucine was added to an ammonium nitrate-maltose medium during cultivation of Streptomyces sp. 732, quinomycin B formation was selectively enhanced (from 3 to 70% of the quinomycin mixture) and quinomycin C synthesis was inhibited completely. In addition, two new quinomycins, designated quinomycins D and E, were produced in the presence of isoleucine. These compounds were found to contain N-methylalloisoleucine. In experiments with isoleucine enantiomorphs, it was determined that the order of effectiveness for quinomycin B synthesis was dl-isoleucine > d-isoleucine > l-isoleucine. The extent to which quinomycin B synthesis is enhanced depends upon the concentration and the time of addition of isoleucine to the medium. The effect of dl-isoleucine was reduced to some extent by the addition of l-valine. It is conceivable that amino acids which are precursors of the N-methylamino acids in quinomycin can regulate quinomycin formation.     

Vitamin B 6 biosynthesis in Bacillus subtilis]

1) A vitamin-B6-producing mutant, BA 1, was selected by treatment of Bacillus subtilis with N-methyl-N'-nitro-N-nitrosoguanidine. Using gradient plates supplemented with the vitamin B6 antagonist isonicotinohydrazide, three mutants of BA 1 were isolated, which excrete 2-5 mg of vitamin B6/l of growth medium. 2) Mutation of the three vitamin-B6-producing strains BA 1, BA 11 and L 71 led to the isolation of 49 vitamin-B6 deficient mutants. All mutants are able to grow with pyridoxine, pyridoxal, pyridoxamine, and even with 4'-deoxypyridoxine. Glycolaldehyde or nicotinic acid do not support growth of the mutants. Some of these vitamin-B6-deficient mutants can also grow in the absence of vitamin B6, providing isoleucine is present. Others show a growth stimulation, when isoleucine is added to a medium containing a vitamin B6 compound. Isoleucine can be replaced by 3-methyl-2-oxovalerate. Cross-feeding experiments indicated a division of the mutants into two groups. Using chromatographic methods, substances which support growth of the mutants were purified, but have not yet been identified. Following the addition of 4'-deoxypyridoxine, 4'-deoxypyridoxine 5'-phosphate was isolated from the growth medium of a vitamin B6-deficient mutant. 3) Threonine dehydratase, transaminase B and transaminase C from wild-type Bacillus subtilis were compared with the enzymes from vitamin-B6-producing strains and with the enzymes from vitamin-B6-deficient mutants. Both the vitamin-B6-producing and the vitamin B6-deficient mutants show higher specific activities than wild type. In the mutant strains no multivalent repression of the threonine dehydratase and transminase B by isoleucine, leucine and valine could be demonstrated. Leucine dehydrogenase, the first enzyme of the isoleucine catabolic pathway, is constitutively produced in the vitamin-B6-producing and in the vitamin-B6-deficient mutants. In the vitamin-B6-deficient mutants, there is a correlation between growth yield in the presence of isoleucine and the specific activity of leucine dehydrogenase. In the crude extract of Bacillus subtilis no pyridoxamine-phosphate oxidase activity could be demonstrated, whereas pyridoxal kinase was readily detectable.     

Role of vitamin B 6 biosynthetic rate in the study of vitamin B 6 synthesis in Escherichia coli

Nutritional auxotrophs of Escherichia coli synthesize vitamin B(6) compounds at a rate of 1 x 10(-10) to 2 x 10(-10) moles per hr per mg (dry weight) of cells when they are suspended in minimal medium lacking their required nutrients. A few auxotrophs have been found to stop or reduce vitamin B(6) synthesis during such an experiment. These include thiamineless, citrate synthaseless, and pyridoxineless mutants as well as mutants which require four carbon compounds for growth. Glycolaldehyde was found to restore vitamin B(6) synthesis in the last named of these mutants without restoring normal growth. A class of pyridoxineless mutants which responded with normal growth to 0.4 mm glycolaldehyde or 0.15 x 10(-3) mm pyridoxol was also found. The results suggest that a thiamine pyrophosphate-requiring step as well as glycolaldehyde may be involved in pyridoxal phosphate biosynthesis.     

家蚕体内因缺乏维生素B6而引起的若干代谢变动

采用不含桑叶粉末、以去维生素牛乳酪蛋白为蛋白源的准合成饲料饲育家蚕Bombyx mori 5龄幼虫,探讨了缺乏维生素B₆（VB₆）对蚕体氨基酸代谢、脂肪酸代谢以及转氨酶活力的影响。缺乏VB₆引起支链氨基酸分解代谢受阻,幼虫体液中大量积累亮氨酸、缬氨酸和异亮氨酸。同时因绢丝腺发育停滞,丝氨酸也在体液中积累。另一方面,缺乏VB₆幼虫体液中赖氨酸、脯氨酸、精氨酸、甲硫氨酸和谷氨酸含量减少,其中赖氨酸尤为突出。推测缺乏VB₆引起赖氨酸分解代谢亢进。结果还表明,缺乏VB₆幼虫体内脂肪酸代谢异常,谷丙转氨酶活力显著低下。     

Biosynthesis of Vitamin B6 by a Yeast Mutant

The gradient-plate technique was employed to isolate mutants of Saccharomyces marxianus (NRRL-Y-1550) which, when grown in a synthetic culture medium, excreted about 2 mug/ml of vitamin B(6) as ascertained by microbiological assay. The major component that possessed vitamin B(6) activity was isolated by ion-exchange column chromatography and identified as pyridoxol by ultraviolet and fluorescence spectroscopy, as well as by paper chromatography and various chemical tests. Pyridoxal was also identified as one of the excreted compounds. Two other compounds that possessed vitamin B(6) activity were excreted in smaller quantities in the growth medium and have not yet been identified; they are not phosphates of vitamin B(6). The amount of vitamin B(6) excreted was not increased when the mutant was grown in the presence of various oxidation products of this vitamin. The methods and results reported here may be helpful in future studies on the biosynthesis of vitamin B(6).     

D- and L-isoleucine metabolism and regulation of their pathways in Pseudomonas putida

Pseudomonas putida oxidized isoleucine to acetyl-coenzyme A (CoA) and propionyl-CoA by a pathway which involved deamination of d-isoleucine by oxidation and l-isoleucine by transamination, oxidative decarboxylation, and beta oxidation at the ethyl side chain. At least three separate inductive events were required to form all of the enzymes of the pathway: d-amino acid dehydrogenase was induced during growth in the presence of d-isoleucine; branched-chain keto dehydrogenase was induced during growth on 2-keto-3-methylvalerate and enzymes specific for isoleucine metabolism; tiglyl-CoA hydrase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase were induced by growth on isoleucine, 2-keto-3-methylvalerate, 2-methylbutyrate, or tiglate. Tiglyl-CoA hydrase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase were purified simultaneously by several enzyme concentration procedures, but were separated by isoelectric focusing. Isoelectric points, pH optima, substrate specificity, and requirements for enzyme action were determined for both enzymes. Evidence was obtained that the dehydrogenase catalyzed the oxidation of 2-methyl-3-hydroxybutyryl-CoA to 2-methylacetoacetyl-CoA. 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase catalyzed the oxidation of 3-hydroxybutyryl-CoA, but l-3-hydroxyacyl-CoA dehydrogenase from pig heart did not catalyze the oxidation of 2-methyl-3-hydroxybutyryl-CoA; therefore, they appeared to be different dehydrogenases. Furthermore, growth on tiglate resulted in the induction of tiglyl-CoA hydrase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase, but these two enzymes were not induced during growth on crotonate or 3-hydroxybutyrate.     

Mutants producing high concentrations of the flavor components active amylalcohol and normal propanol in Saccharomyces cerevisiae

Mutants resistant to the isoleucine analogue dl-thiaisoleucine (TIL) which produced large amounts of the flavor components active amylalcohol and normal propanol were isolated from sake, and baker's and laboratory yeasts Saccharomyces cerevisiae. These mutants had threonine deaminase (EC 4.2.1.16) with decreased feedback sensitivity to l-isoleucine and accumulated a high concentration of isoleucine as well as these higher alcohols. Genetic analysis using a laboratory strain revealed that the mutation for increased production of active amylalcohol and normal propanol was controlled by a single dominant gene, and evidence was found for linkage between TIL-resistance and the increased production ability of these higher alcohols. Sake and bread made with the mutants contained more of these components and had enhanced flavor.     

Isoleucine and valine metabolism of Escherichia coli. XIV. Effect of thiaisoleucine

Thiaisoleucine (2-amino-3-methylthiobutyrate) completely inhibited the growth of strain K-12 of Escherichia coli at a concentration of 5 x 10(-3)m. The inhibition was antagonized by growth-factor amounts of l-isoleucine. Thiaisoleucine inhibited the deamination of threonine and the transfer of (14)C-isoleucine to soluble ribonucleic acid and underwent transamination with alpha-ketoglutarate as the amino acceptor. In each case, the analogue appeared to be less effective than isoleucine as either an inhibitor or substrate.     

Isoleucine auxotrophy as a consequence of a mutationally altered isoleucyl-transfer ribonucleic acid synthetase

Among mutants which require isoleucine, but not valine, for growth, we have found two distinguishable classes. One is defective in the biosynthetic enzyme threonine deaminase (l-threonine hydro-lyase, deaminating, EC 4.2.1.16) and the other has an altered isoleucyl transfer ribonucleic acid (tRNA) synthetase [l-isoleucine: soluble RNA ligase (adenosine monophosphate), EC 6.1.1.5]. The mutation which affects ileS, the structural gene for isoleucyl-tRNA synthetase, is located between thr and pyrA at 0 min on the map of the Escherichia coli chromosome. This mutationally altered isoleucyl-tRNA synthetase has an apparent K(m) for isoleucine ( approximately 1 mm) 300-fold higher than that of the enzyme from wild type; on the other hand, the apparent V(max) is altered only slightly. When the mutationally altered ileS allele was introduced into a strain which overproduces isoleucine, the resulting strain could grow without addition of isoleucine. We conclude that the normal intracellular isoleucine level is not high enough to allow efficient charging to tRNA(Ile) by the mutant enzyme because of the K(m) defect. A consequence of the alteration in isoleucyl-tRNA synthetase was a fourfold derepression of the enzymes responsible for isoleucine biosynthesis. Thus, a functional isoleucyl-tRNA synthetase is needed for isoleucine to act as a regulator of its own biosynthesis.     

Mechanism and Regulation of Isoleucine Excretion in Corynebacterium glutamicum

Whole cells of Corynebacterium glutamicum were loaded with high cytoplasmic l-isoleucine concentrations, and isoleucine excretion from these cells was studied in terms of mechanism and regulation. The transmembrane isoleucine flux could be differentiated into carrier-mediated uptake, carrier-mediated excretion, and diffusion. After discrimination from the other transmembrane solute movements, the outward-directed flux, which was due to the activity of the isoleucine excretion carrier, was characterized with respect to its energy dependence and its regulation at the level of expression. Isoleucine excretion was shown to function as a secondary transport process, driven by the membrane potential and coupled to the movement of protons, presumably with a stoichiometry of 2:1 (H(sup+)/isoleucine). Of a variety of putative transport substrates, only leucine was able to compete for isoleucine at the cis (cytosolic) side of the export carrier. Cytoplasmic isoleucine concentrations higher than 20 mM induce the activity of the isoleucine excretion system. This effect is specific for isoleucine and is inhibited by the presence of chloramphenicol. Apart from leucine, other amino acids and related amino acid analogs are not able to induce isoleucine excretion. The complex pattern of regulation of the isoleucine excretion system at the level of activity and expression is shown to be related to the pattern of regulation of the isoleucine uptake system in C. glutamicum in terms of physiological significance.     

Stereoselective incorporation of isoleucine into Cypridina luciferin in Cypridina hilgendorfii (Vargula hilgendorfii)

The emission of light in the marine ostracod Cypridina hilgendorfii (presently Vargula hilgendorfii) is produced by the Cypridina luciferin-luciferase reaction in the presence of molecular oxygen. Cypridina luciferin has an asymmetric carbon derived from isoleucine, and the absolute configuration is identical to the C-3 position in L-isoleucine or D-alloisoleucine. To determine the stereoselective incorporation of the isoleucine isomers (L-isoleucine, D-isoleucine, L-alloisoleucine, and D-alloisoleucine), we synthesized four (2)H-labeled isoleucine isomers and examined their incorporation into Cypridina luciferin by feeding experiments. Judging by these results, L-isoleucine is predominantly incorporated into Cypridina luciferin. This suggests that the isoleucine unit of Cypridina luciferin is derived from L-isoleucine, but not from D-alloisoleucine.     

Isolation of four isomers of the 20,000 dalton variant of human pituitary growth hormone

A simple procedure has been developed which for the first time describes the isolation of isomers of the 20,000 dalton variant of human growth hormone (20K hGH). From a human pituitary hormone concentrate different hGH dimers (covalently and noncovalently linked) were enriched by chromatography on SP-Sephadex C-50, DEAE-Sepharose CL-6B and Sephadex G-100. Noncovalently-linked dimers were split by 6 M urea into 20K hGH and 22K hGH monomers. A complete group-separation of 20K hGH and 22K hGH monomers was achieved by chromatography on DEAE-Sepharose CL-6B at neutral pH. The 20K hGH monomer was resolved into four isomers either by preparative isoelectric focusing or by zone electrophoresis in agarose suspension at alkaline pH. The three latter techniques were all used in the presence of 6 M urea. Radioimmunoassay and radioreceptorassay indicated that the isomers obtained were true components of human growth hormone.     

Microbial production of vitamin B12 antimetabolites: II. 2-Amino-4-keto-3-methylpentanoic acids from bacillus cereus 439

2-Amino-4-keto-3-methylpentanoic acids were isolated as a diastereomeric mixture from Bacillus cereus 439 fermentations and found to be vitamin B₁₂ antimetabolites in a bioassay system based on the vitamin B₁₂-requiring Escherichia coli (Davis 113-3). A similar diastereomeric mixture with bioactivity was synthesized by condensation of 2-bromo-3-butanone with sodio diethyl acetamidomalonate followed by hydrolysis with 6 N HCl and purification by ion-exchange chromatography. The growth inhibitory effects of the antimetabolite were reversed by vitamin B₁₂, l-methionine, l-isoleucine, l-leucine, l-valine, and d-alanine.     

Isolation of Four Isomers of the 20,000 Dalton Variant of Human Pituitary Growth Hormone

A simple procedure has been developed which for the first time describes the isolation of isomers of the 20, 000 dalton variant of human growth hormone (20K hGH). From a human pituitary hormone concentrate different hGH dimers (covalently and noncova-lently linked) were enriched by chromatography on SP-Sephadex C-50, DEAE-Sepharose CL-6B and Sephadex G-100. Noncovalently-1inked dimers were split by 6 M urea into 20K hGH and 22K hGH monomers. A complete group-separation of 20K hGH and 22K hGH monomers was achieved by chromatography on DEAE-Sepharose CL-6B at neutral pH. The 20K hGH monomer was resolved into four isomers either by preparative isoelectric focusing or by zone electrophoresis in agarose suspension at alkaline pH. The three latter techniques were all used in the presence of 6 M urea. Radioimmunoassay and radioreceptorassay indicated that the isomers obtained were true components of human growth hormone.     

Use of Feedback-Resistant Threonine Dehydratases of Corynebacterium glutamicum To Increase Carbon Flux towards l-Isoleucine

The biosynthesis of l-isoleucine proceeds via a highly regulated reaction sequence connected with l-lysine and l-threonine synthesis. Using defined genetic Corynebacterium glutamicum strains characterized by different fluxes through the homoserine dehydrogenase reaction, we analyzed the influence of four different ilvA alleles (encoding threonine dehydratase) in vectors with two different copy numbers on the total flux towards l-isoleucine. For this purpose, 18 different strains were constructed and analyzed. The result was that unlike ilvA in vectors with low copy numbers, ilvA in high-copy-number vectors increased the final l-isoleucine yield by about 20%. An additional 40% increase in l-isoleucine yield was obtained by the use of ilvA alleles encoding feedback-resistant threonine dehydratases. The strain with the highest yield was characterized by three hom(Fbr) copies encoding feedback-resistant homoserine dehydrogenase and ilvA(Fbr) encoding feedback-resistant threonine dehydratase on a multicopy plasmid. It accumulated 96 mM l-isoleucine, without any l-threonine as a by-product. The highest specific productivity was 0.052 g of l-isoleucine per g of biomass per h. This comparative flux analysis of isogenic strains showed that high levels of l-isoleucine formation from glucose can be achieved by the appropriate balance of homoserine dehydrogenase and threonine dehydratase activities in a strain background with feedback-resistant aspartate kinase. However, still-unknown limitations are present within the entire reaction sequence.     

Comparative studies on the microbiological vitamin B12 assay at two laboratories.

The turbidimetric methods in routine use at two laboratories for the microbiological assay of vitamin B12 have been compared. Attempts were made to standardize some major parts of the method, i.e., assay design, test strain (Lactobacillus leichmannii), test medium, and reference standard. The laboratories used different approaches to achieve efficient assay procedures. During a 6-year period four comparative experiments were carried out. In these experiments the vitamin B12 content of five different products was determined in a series of independent assays at each laboratory. A satisfactory degree of agreement (difference less than 5%) was found for four of these products.     

Comparative studies on the microbiological vitamin B12 assay at two laboratories.

The turbidimetric methods in routine use at two laboratories for the microbiological assay of vitamin B12 have been compared. Attempts were made to standardize some major parts of the method, i.e., assay design, test strain (Lactobacillus leichmannii), test medium, and reference standard. The laboratories used different approaches to achieve efficient assay procedures. During a 6-year period four comparative experiments were carried out. In these experiments the vitamin B12 content of five different products was determined in a series of independent assays at each laboratory. A satisfactory degree of agreement (difference less than 5%) was found for four of these products.     

Bacterial Utilization of Dodecyl Sulfate and Dodecyl Benzene Sulfonate

Two unknown bacterial isolants (C12 and C12B) were obtained from enriched soils and cultured on media containing detergent compounds as sole sources of carbon. Either isolant destroyed the foaming capacity of cultures containing dodecyl sulfate; but C12B, which could grow on dodecyl benzene sulfonate (DBS) whereas C12 could not, did not destroy the foaming capacity of this surfactant. The source of DBS available in quantity was a mixture of isomers derived from kerosene, and the bacteria utilized only one-fifth to one-fourth of this material during growth. Both isolants grew on short- or long-chained organic acids, and resting cells of both rapidly oxidized several long-chain acids and alcohols. Three of five phenyl-placement isomers of DBS (with the phenyl group at carbon 2, 3, or 6 on the alkyl chains) were excellent substrates for growth of C12B, but isomers with phenyl placement at carbon 4 or 5 were toxic and killed the bacteria.     

Vitamin B6 compounds prevent the death of yeast cells due to menadione, a reactive oxygen generator

The antioxidant effects of natural vitamin B(6) compounds on Schizosaccharomyces pombe cells treated with menadione sodium bisulfite (water-soluble menadione and a generator of superoxide, MSB) and the mechanism underlying the function were examined with the yeast cells treated with pyridoxal 5'-phosphate. Vitamin B(6) compounds showed no ex vivo reactivity toward MBS at pH 5.5 or 7.0. The yeast cells showed no growth in the medium containing 1.0 mM MSB. The coexistence of 1.0 mM of each vitamin B(6) compound supported the growth of the yeast cells. The efficacy order was pyridoxal 5'-phosphate>/=pyridoxamine 5'-phosphate>pyridoxamine>pyridoxal>/=pyridoxine. The first three compounds showed higher antioxidant activity than vitamin C did. Pyridoxal 5'-phosphate prevented the reduction of the glutathione content in the MSB-treated cells and, in turn, suppressed the increases in peroxide and thiobarbituric acid reactive substances in the yeast cells and increased the viability of the yeast cells under oxidative stress. The antioxidant function of pyridoxal 5'-phosphate was not dependent on the phosphorelay pathway, which finally triggers the expression of the catalase gene.     

Cysteine and growth inhibition of Escherichia coli: threonine deaminase as the target enzyme.

Cysteine has been shown to inhibit growth in Escherichia coli strains C6 and HfrH 72, but not M108A. Growth inhibition was overcome by inclusion of isoleucine, leucine, and valine in the medium. Isoleucine biosynthesis was apparently affected, since addition of this amino acid alone could alter the inhibitory effects of cysteine. Homocysteine, mercaptoethylamine, and mercaptoethanol inhibited growth to varying degrees in some strains, these effects also being prevented by addition of branched-chain amino acids. Cysteine, mercaptoethylamine, and homocysteine were inhibitors of threonine deaminase but not transaminase B, two enzymes of the ilvEDA operon. Cysteine inhibition of threonine deaminase was reversed by threonine, although the pattern of inhibition was mixed. These results suggest a relationship between the growth-inhibitory effects of cysteine and other sulfur compounds and the inhibition of isoleucine synthesis at the level of threonine deaminase.