Purification and characterization of a dehydrogenase catalyzing conversion of N alpha-benzyloxycarbonyl-L-aminoadipic-delta-semialdehyde to N alpha-benzyloxycarbonyl-L-aminoadipic acid from rhodococcus sp. AIU Z-35-1.

The enzyme catalyzing conversion of N alpha-benzyloxycarbonyl-L-aminoadipic-delta-semialdehyde (N alpha-Z-L-AASA) to N alpha-benzyloxycarbonyl-L-aminoadipic acid (N alpha-Z-L-AAA) in Rhodococcus sp. AIU Z-35-1 was identified, and its characteristics were revealed. This reaction was catalyzed by a dehydrogenase with a molecular mass of 59 kDa. The dehydrogenase exhibited enzyme activity on not only N alpha-Z-L-AASA but also N alpha-Z-D-AASA and short chain aliphatic aldehydes, but not on aromatic aldehydes and alcohols. The apparent Km values for N alpha-Z-L-AASA, N alpha-Z-D-AASA and NAD+ were estimated to be 3.8 mM, 14.1 mM and 0.16 mM, respectively. The NH2 terminal amino acid sequence of this enzyme exhibited a similarity to those of a piperidein-6-carboxylate dehydrogenase from Streptomyces clavuligerus and a putative dehydrogenase from Rhodococcus sp. RHA 1, but not to those of other microbial aldehyde dehydrogenases.

A new microbial method for more efficient production of Nalpha-benzyloxycarbonyl-L-aminoadipate delta-semialdehyde and Nalpha-benzyloxycarbonyl-D-aminoadipate delta-semialdehyde.

A new biochemical method for more efficient production of Nalpha-benzyloxycarbonyl-L-aminoadipate delta-semialdehyde (Nalpha-Z-L-AASA) and Nalpha-Z-D-AASA was developed with cells of Rhodococcus sp. AIU Z-35-1. Using the cells harvested after 1 d of cultivation, more than 95 mM Nalpha-Z-L-AASA was produced from 100 mM Nalpha-Z-L-lysine by incubating at pH 5.0 for 1 d at 30 degrees C or by incubating at pH 7.0 for 2 d at 10 degrees C. A similar conversion yield of Nalpha-Z-D-AASA was also obtained under the same conditions. These reaction times required were 1/4 and 1/2 of the respective ones by the method with amine oxidase, and the yields of Nalpha-Z-L-AASA and Nalpha-Z-D-AASA were 2 times higher than the respective ones by the method with amine oxidase. In addition, this method had the advantages of not requiring purification of enzyme and addition of catalase. Thus, the microbial method proposed here was superior to the chemical and other biochemical methods in simplicity, reaction rate, and yield.

Characterization of the oat1 gene of Penicillium chrysogenum encoding an omega-aminotransferase: induction by L-lysine, L-ornithine and L-arginine and repression by ammonium.

The Penicillium chrysogenum oat1 gene, which encodes a class III omega-aminotransferase, was cloned and characterized. This enzyme converts lysine into 2-aminoadipic semialdehyde, and plays an important role in the biosynthesis of 2-aminoadipic acid, a precursor of penicillin and other beta-lactam antibiotics. The enzyme is related to ornithine-5-aminotransferases and to the lysine-6-aminotransferases encoded by the lat genes found in bacterial cephamycin gene clusters. Expression of oat1 is induced by lysine, ornithine and arginine, and repressed by ammonium ions. AreA-binding GATA and GATT sequences involved in regulation by ammonium, and an 8-bp direct repeat associated with arginine induction in Emericella (Aspergillus nidulans and Saccharomyces cerevisiae, were found in the oat1 promoter region. Deletion of the oat1 gene resulted in the loss of omega-aminotransferase activity. The null mutants were unable to grow on ornithine or arginine as sole nitrogen sources and showed reduced growth on lysine. Complementation of the null mutant with the oat1 gene restored normal levels of omega-aminotransferase activity and the ability to grow on ornithine, arginine and lysine. The role of the oat1 gene in the biosynthesis of 2-aminoadipic acid is discussed.

In Saccharomyces cerevisae, feedback inhibition of homocitrate synthase isoenzymes by lysine modulates the activation of LYS gene expression by Lys14p.

Expression of the structural genes for lysine biosynthesis responds to an induction mechanism mediated by the transcriptional activator Lys14p in the presence of alpha-aminoadipate semialdehyde (alphaAASA), an intermediate of the pathway acting as a coinducer. This activation is reduced by the presence of lysine in the growth medium, leading to apparent repression. In this report we demonstrate that Saccharomyces cerevisiae possesses two genes, LYS20 and LYS21, encoding two homocitrate synthase isoenzymes which are located in the nucleus. Each isoform is inhibited by lysine with a different sensitivity. Lysine-overproducing mutants were isolated as resistant to aminoethylcysteine, a toxic lysine analog. Mutations, LYS20fbr and LYS21fbr, are allelic to LYS20 and LYS21, and lead to desensitization of homocitrate synthase activity towards lysine and to a loss of apparent repression by this amino acid. There is a fair correlation between the I0.5 of homocitrate synthase for lysine, the intracellular lysine pool and the levels of Lys enzymes, confirming the importance of the activity control of the first step of the pathway for the expression of LYS genes. The data are consistent with the conclusion that inhibition by lysine of Lys14p activation results from the control of alphaAASA production through the feedback inhibition of homocitrate synthase activity.

The pcd gene encoding piperideine-6-carboxylate dehydrogenase involved in biosynthesis of alpha-aminoadipic acid is located in the cephamycin cluster of Streptomyces clavuligerus.

Three open reading frames (ORFs) have been located downstream of cefE in the cephamycin C gene cluster of Streptomyces clavuligerus. ORF13 (pcd) encodes a 496-amino-acid protein (molecular weight [MW], 52,488) with an N-terminal amino acid sequence identical to that of pure piperideine-6-carboxylate dehydrogenase. ORF14 (cmcT) encodes a 523-amino-acid protein (MW, 54,232) analogous to Streptomyces proteins for efflux and resistance to antibiotics. ORF15 (pbp74) encodes a high molecular weight penicillin-binding protein (MW, 74, 094).

Delta-1-piperideine-6-carboxylate dehydrogenase, a new enzyme that forms alpha-aminoadipate in Streptomyces clavuligerus and other cephamycin C-producing actinomycetes.

Delta-1-Piperideine-6-carboxylate (P6C) dehydrogenase activity, which catalyses the conversion of P6C into alpha-aminoadipic acid, has been studied in the cephamycin C producer Streptomyces clavuligerus by both spectrophotometric and radiometric assays. The enzyme has been purified 124-fold to electrophoretic homogeneity with a 26% yield. The native protein is a monomer of 56.2 kDa that efficiently uses P6C (apparent Km 14 microM) and NAD+ (apparent Km 115 microM), but not NADP+ or other electron acceptors, as substrates. The enzyme activity was inhibited (by 66%) by its end product NADH at 0.1 mM concentration. It did not show activity towards pyrroline-5-carboxylate and was separated by Blue-Sepharose chromatography from pyrroline-5-carboxylate dehydrogenase, an enzyme involved in the catabolism of proline. P6C dehydrogenase reached maximal activity later than other early enzymes of the cephamycin pathway. The P6C dehydrogenase activity was decreased in ammonium (40 mM)-supplemented cultures, as was that of lysine 6 amino-transferase. P6C dehydrogenase activity was also found in other cephamycin C producers (Streptomyces cattleya and Nocardia lactamdurans) but no in actinomycetes that do no produce beta-lactams, suggesting that it is an enzyme specific for cephamycin biosynthesis, involved in the second stage of the two-step conversion of lysine to alpha-aminoadipic acid.

Subcellular compartmentation of penicillin biosynthesis in Penicillium chrysogenum. The amino acid precursors are derived from the vacuole.

The cellular localization of the origin of alpha-aminoadipate used in penicillin biosynthesis and the first enzymic step in Penicillium chrysogenum involved, delta-(alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), has been studied. Subcellular fractions were obtained from protoplasts of a high penicillin-producing strain upon lysis by Triton X-100, and vacuoles purified from them. They were identified by the aid of alpha-mannosidase as a marker enzyme, by the presence of polyphosphate, and their ability to sequester [14C]lysin, added to the protoplasts prior to subcellular fractionation. 15.6 and 26.5%, respectively, of 6-[14C]alpha-aminoadipate, and 8.5 and 10.3%, respectively, of [14C]valine added accordingly were also found in the vacuole, and the higher proportion was found in vacuoles isolated from penicillin-producing mycelia. ACVS protein was detected in the membrane as well as the soluble fraction of the purified vacuoles. We propose therefore that ACVS is located either within or bound to the vacuolar membrane, and that the precursor amino acids for penicillin biosynthesis are withdrawn from the vacuolar amino acid pool.

Enzymatic production of alpha-aminoadipate-delta-semialdehyde and related compounds by lysine epsilon-dehydrogenase from Candida albicans.

L-Lysine epsilon-dehydrogenase [L-lysine:NADP+ oxidoreductase (epsilon-deaminating), EC 1.4.1.15] of Candida albicans was studied emphasizing its application for the production of alpha-aminoadipate-delta-semialdehyde and related compounds. A high enzyme level (240 pkat/mg of protein in the crude extract) could be attained during growth in the presence of L-lysine as sole nitrogen source. After optimization of the reaction conditions a partial purified enzyme (1.5 nkat/mg of protein) was used to produce alpha-aminoadipate-delta-semialdehyde, S-(beta-acetaldehyde)-cysteine, alpha-amino-delta-hydroxyadipate-semialdehyde and alpha-amino-gamma-hydroxyadipate-semialdehyde from the corresponding substrates lysine, S-(beta-aminoethyl)-cysteine, 5-hydroxylysine and 4-hydroxylysine, respectively. After purification of the compounds using Dowex 50 x 4 chromatography a yield of the products between 4.6 and 6.8% was achieved.

Lysine catabolism in Streptomyces spp. is primarily through cadaverine: beta-lactam producers also make alpha-aminoadipate.

Genetic and biochemical evidence was obtained for lysine catabolism via cadaverine and delta-aminovalerate in both the beta-lactam producer Streptomyces clavuligerus and the nonproducer Streptomyces lividans. This pathway is used when lysine is supplied as the sole source of nitrogen for the organism. A second pathway for lysine catabolism is present in S. clavuligerus but not in S. lividans. It leads to alpha-aminoadipate, a precursor for beta-lactam biosynthesis. Since it does not allow S. clavuligerus to grow on lysine as the sole nitrogen source, this pathway may be used exclusively to provide a precursor for beta-lactam biosynthesis. beta-Lactam producers were unable to grow well on alpha-aminoadipate as the only nitrogen source, whereas three of seven species not known to produce beta-lactam grew well under the same conditions. Lysine epsilon-aminotransferase, the initial enzyme in the alpha-aminoadipate pathway for lysine catabolism, was detected in cell extracts only from the beta-lactam producers. These results suggest that synthesis of alpha-aminoadipate is exclusively a secondary metabolic trait, present or expressed only in beta-lactam producers, while genes governing the catabolism of alpha-aminoadipate are present or fully expressed only in beta-lactam nonproducers.

L-lysine epsilon-aminotransferase involved in cephamycin C synthesis in Streptomyces lactamdurans.

In Streptomyces lactamdurans, the precursor of the alpha-aminoadipoyl side-chain of cephamycin C is L-lysine. In this regard, streptomycetes differ strikingly from the fungi, which produce alpha-aminoadipic acid during the synthesis, rather than the breakdown, of L-lysine. Studies using a cell-free system showed that an aminoadipic acid. The product of this reaction was trapped and subsequently purified by ion-exchange chromatography. Thin-layer chromatography, spectrophotometry, and amino acid oxidase digestion studies identified the reaction product as L-1-piperideine-6-carboxylate, implying enzymatic removal of the epsilon amino group of L-lysine. This enzymatic activity (E.C. 2.6.1.36; L-lysine: 2-oxoglutarate 6-aminotransferase) is highly unusual and was previously conclusively demonstrated only in the genus Flavobacterium. In S. lactamdurans, the specific activity of this enzyme reaches a peak early in the fermentation (approximately 20 h) and decreases as the antibiotic begins to appear.

A simple method for the preparation of D-alpha-aminoadipic acid.

High quantity (1 g and more) of racemically and chromatographically pure D-alpha-aminoadipic acid was prepared by selective metabolism of the L-isomer of the commercially available DL-alpha-aminoadipate by Pseudomonas putida. The overall yield of this preparation averaged 40%. The final product has [a]25D value of -25 degrees. This procedure can be useful in the synthesis of high purity D-alpha-amino-adipate, a compound shown recently to be a useful tool in the study of neurotransmission mechanism mediating synaptic excitation.

Effect of vitamin B6 deficiency on collagen metabolism in rats.

The effects of VB6 deficiency on collagen metabolism were studied using young and adult rats. It was observed in VB6 deficient rats, that both the amount of urinary hydroxyproline and salt soluble fraction of skin collagen decreased. Furthermore, decrease of the alpha-component and increase of the beta-component were also found in the soluble collagen. However, the activity of plasma amine oxidase was lower and the aldehyde content in the acid soluble collagen were also lower than the control group. These changes were revealed more clearly in young growing rats than in adult rats. Based on these results, it may be that VB6 participates in the allysine (alpha-amino-adipic-delta-semialdehyde) formation which is considered as the first step of collagen maturation and also in the synthesis of protocollagen peptide chains.