Properties and interaction of heterologously expressed glutamate decarboxylase isoenzymes GAD(65kDa) and GAD(67kDa) from human brain with ginkgotoxin and its 5'-phosphate.

Two isoforms of glutamate decarboxylase (GAD(65kDa) and GAD(67kDa)) from human brain, which had previously been overexpressed in Escherichia coli as fusion proteins containing a glutathione-S-transferase domain, were purified by affinity chromatography on glutathione Sepharose 4B. Both isoforms were also expressed in Saccharomyces cerevisiae. After modification of a HPLC based assay, the enzymes were characterized with respect to their biochemical properties. Comparison of kinetic data, pH, and temperature optima as well as of the mode of interaction with pyridoxal phosphate as a cofactor revealed several significant differences between the two isoenzymes reflecting their somewhat different physiological and molecular features. Investigation of the influence of 4'-O-methylpyridoxine (ginkgotoxin) (1), a neurotoxin occurring in Ginkgo biloba L., on the different isoenzymes, indicates that the phosphorylated form of the toxin, 4'-O-methylpyridoxine-5'-phosphate (2), decreases GAD(65kDa) activity, although in unphysiologically high concentrations, whereas GAD(67kDa) activity seems to be hardly affected.

Mutational analysis and reconstituted expression of the biosynthetic genes involved in the formation of 3-amino-5-hydroxybenzoic acid, the starter unit of rifamycin biosynthesis in amycolatopsis Mediterranei S699.

To investigate a novel branch of the shikimate biosynthesis pathway operating in the formation of 3-amino-5-hydroxybenzoic acid (AHBA), the unique biosynthetic precursor of rifamycin and related ansamycins, a series of target-directed mutations and heterologous gene expressions were investigated in Amycolatopsis mediterranei and Streptomyces coelicolor. The genes involved in AHBA formation were inactivated individually, and the resulting mutants were further examined by incubating the cell-free extracts with known intermediates of the pathway and analyzing for AHBA formation. The rifL, -M, and -N genes were shown to be involved in the step(s) from either phosphoenolpyruvate/d-erythrose 4-phosphate or other precursors to 3,4-dideoxy-4-amino-d-arabino-heptulosonate 7-phosphate. The gene products of the rifH, -G, and -J genes resemble enzymes involved in the shikimate biosynthesis pathway (August, P. R., Tang, L., Yoon, Y. J., Ning, S., Müller, R., Yu, T.-W., Taylor, M., Hoffmann, D., Kim, C.-G., Zhang, X., Hutchinson, C. R., and Floss, H. G. (1998) Chem. Biol. 5, 69-79). Mutants of the rifH and -J genes produced rifamycin B at 1% and 10%, respectively, of the yields of the wild type; inactivation of the rifG gene did not affect rifamycin production significantly. Finally, coexpressing the rifG-N and -J genes in S. coelicolor YU105 under the control of the act promoter led to significant production of AHBA in the fermented cultures, confirming that seven of these genes are indeed necessary and sufficient for AHBA formation. The effects of deletion of individual genes from the heterologous expression cassette on AHBA formation duplicated the effects of the genomic rifG-N and -J mutations on rifamycin production, indicating that all these genes encode proteins with catalytic rather than regulatory functions in AHBA formation for rifamycin biosynthesis by A. mediterranei.

Biosynthesis of vitamin B6 and structurally related derivatives.

In spite of the rather simple structure of pyridoxal 5'-phosphate (I), a member of the vitamin B6 group, the elucidation of its de novo biosynthesis remained largely unexplored until recently. Experiments designed to investigate the formation of the vitamin B6 pyridine nucleus mainly concentrated on Escherichia coli. The results of tracer experiments with radioactive and stable isotopes, feeding experiments, and molecular biological studies led to the prediction that 4-hydroxy-L-threonine (VIII, R = H) and 1-deoxy-D-xylulose (VII, R = H) are precursors which are assembled to yield the carbon-nitrogen skeleton of vitamin B6. At this point, the involvement of the phosphorylated forms of these precursors in this assembly seems quite clear. However, vitamin B6 biosynthesis in organisms other than E. coli remains largely unknown. Toxic derivatives of vitamin B6, such as ginkgotoxin, occurring in higher plants may be suitable targets to gain further insight into this tricky problem.

Clustering of isochorismate synthase genes menF and entC and channeling of isochorismate in Escherichia coli.

There are two isochorismate synthase genes entC and menF in Escherichia coli. They encode enzymes (isochorismate synthase, EC 5.4.99.6) which reversibly synthesize isochorismic acid from chorismic acid. The genes share a 24.2% identity but are differently regulated. Activity of the MenF isochorismate synthase is significantly increased under anaerobic conditions whereas the activity of the EntC isochorismate synthase is greatly stimulated during growth in an iron deficient medium. Isochorismic acid synthesized by EntC is mainly channeled into enterobactin synthesis whereas isochorismic acid synthesized by MenF is mainly channeled into menaquinone synthesis. When menF or entC were separately placed onto overexpression plasmids and the plasmids introduced into a menF(-)/entC(-) double mutant in two separate experiments, the isochorismate formed was fed into both, the menaquinone and the enterobactin pathway. Moreover, in spite of a high isochorismate synthase activity menaquinone and enterobactin formation were not fully restored, indicating that isochorismate was lost by diffusion. Thus, under these conditions channeling was not observed. We conclude that in E. coli the chromosomal position of both menF and entC in their respective clusters is a prerequisite for channeling of isochorismate in both pathways.

Biosynthesis of 4'-O-methylpyridoxine (Ginkgotoxin) from primary precursors.

Cell suspension cultures of Ginkgo biloba and Albizia tanganyicensis were investigated for the presence of 4'-O-methylpyridoxine (ginkgotoxin, 2), the 4'-O-methyl derivative of vitamin B(6) (pyridoxine, 1). The cultures produced the toxin even in the absence of vitamin B(6) (a common additive to plant cell culture media). This indicates that the pyridoxine ring system of ginkgotoxin is synthesized de novo by the cultured cells. A feeding experiment with D-[U-(13)C(6)]glucose revealed that the mode of incorporation of label into the pyridoxine moiety of 2 matched that observed for 1 in Escherichia coli. Thus, the data obtained in this investigation provide independent proof supporting the current hypothesis on vitamin B(6) biosynthesis. The 4'-O-methyl group of ginkgotoxin (2) was labeled from L-[methyl-(13)C(1)]methionine. This indicates that ginkgotoxin is likely to be derived by 4'-O-methylation of pyridoxine (1). The G. biloba cell suspension culture may be a suitable system to get further insight into vitamin B(6) and/or ginkgotoxin biosynthesis.

Biosynthesis of ansatrienin (mycotrienin) and naphthomycin. Identification and analysis of two separate biosynthetic gene clusters in Streptomyces collinus Tü 1892.

The polyketide chains of the two ansamycin antibiotics, ansatrienin (mycotrienin) and naphthomycin produced by Streptomyces collinus are assembled using 3-amino-5-hydroxybenzoic acid (AHBA) as a starter unit. The gene encoding AHBA synthase, an enzyme which catalyzes the final step of AHBA biosynthesis in the recently discovered aminoshikimate pathway, has been used to identify two separate antibiotic biosynthetic gene clusters in S. collinus. In one of these clusters, analysis of approximately 20 kb of contiguous sequence has revealed both a cluster of six genes presumed to play a role in the AHBA pathway and the beginning of a polyketide synthase (PKS) gene containing an acyl ACP ligase domain. This domain is likely responsible for loading AHBA onto the PKS. This gene cluster also contains chcA, encoding the enzyme 1-cyclohexenylcarbonyl CoA reductase, which is essential for the biosynthesis of the cyclohexanecarboxylic acid moiety of ansatrienin from shikimic acid, and a peptide synthetase. This gene cluster thus seems to control the biosynthesis of ansatrienin, which contains a side chain of N-cyclohexanecarbonyl-d-alanine esterified to the macrocyclic lactam backbone. In the putative naphthomycin biosynthetic gene cluster approximately 13 kb of contiguous sequence has revealed a second set of the genes required for AHBA biosynthesis. In addition the end of a polyketide synthase and a gene putatively involved in termination of the chain extension process, formation of an intramolecular amide bond between the AHBA nitrogen and the carboxyl group of the fully extended polyketide chain, have been identified. Thus, despite commonality in biosynthesis, the ansatrienin and naphthomycin biosynthetic gene clusters show clear organizational differences and carry separate sets of genes for AHBA biosynthesis.

The role of isochorismate hydroxymutase genes entC and menF in enterobactin and menaquinone biosynthesis in Escherichia coli.

Klebsiella pneumoniae 62-1, a triple mutant impaired in aromatic amino acid biosynthesis (Phe-, Tyr-, Trp-), excretes chorismic acid into the culture broth. When transformed with plasmids harbouring Escherichia coli genes entC or menF the mutant excretes a mixture of both chorismic and isochorismic acid indicating that not only entC but also menF encodes an isochorismate hydroxymutase (isochorismate synthase, EC 5.4.99.6) enzyme. These enzymes catalyze the first step in enterobactin or menaquinone biosynthesis, respectively. Although both gene products (EntC and MenF) catalyze the same reaction, they play distinct roles in the biosynthesis of menaquinone (MK8) and enterobactin. An E. coli mutant (PBB7) with an intact menF but a disrupted entC produced menaquinone (MK8) but no enterobactin, whereas a mutant (PBB9) with an intact entC but a disrupted menF produced enterobactin and only a trace of menaquinone (MK8). When both menF and entC were disrupted (mutant PBB8) neither menaquinone (MK8) nor enterobactin was detectable. Our previous assumption that entC is responsible for both menaquinone and enterobactin biosynthesis is inconsistent with these mutant studies and has to be revised. The presence in the promoter region of menF of a putative cAMP receptor protein binding site indicates that menF is regulated differently from entC. The menF gene was overexpressed as a fusion gene and its product (6xHis-tagged MenF) isolated. The enzyme catalyzed the formation of isochorismic from chorismic acid and as opposed to a previous publication also the reverse reaction. The enzyme was characterized and its kinetic data determined.

Occurrence of neurotoxic 4'-O-methylpyridoxine in Ginkgo biloba leaves, Ginkgo medications and Japanese Ginkgo food.

4'-O-methylpyridoxine (ginkgotoxin) is a neurotoxic antivitamin B6 which occurs in Ginkgo biloba L. seeds. Contrary to a previous report by Wada et al., the toxin was also detected in Ginkgo biloba leaves. The leaves are a source of extracts (e.g. EGb761) employed in the preparation of Ginkgo medications. Consequently the toxin is also present in Ginkgo medications and is even detectable in homoeopathic preparations. The toxin occurs also in boiled Japanese Ginkgo food. However, the amount of the toxin is likely to be too low to exert a detrimental effect after administration of the medication or ingestion of food.

4-O-phosphoryl-L-threonine, a substrate of the pdxC(serC) gene product involved in vitamin B6 biosynthesis.

The Escherichia coli pdxC(serC) gene codes for a transaminase (EC 2.6.1.52). The gene is involved in both pyridoxine (vitamin B6) and serine biosynthesis and was overexpressed as a MalE/PdxC(SerC) fusion protein. The fusion protein was purified by affinity chromatography on an amylose resin and hydrolyzed in the presence of protease factor Xa. Both the fusion protein and the PdxC(SerC) protein were characterized (K(M) value, turnover number, optimum pH). Both enzymes used 4-O-phosphoryl-L-threonine rather than 4-hydroxy-L-threonine as a substrate indicating that the phosphorylated rather than the non-phosphorylated amino acid is involved in pyridoxine biosynthesis. Pyridoxal phosphate was shown to be the cofactor for both enzymes and therefore seems to be involved in its own biosynthesis.

An isochorismate hydroxymutase isogene in Escherichia coli.

The pivotal step in enterobactin and menaquinone biosynthesis is the conversion of chorismate to isochorismate. Circumstantial evidence pointed to Escherichia coli isochorismate hydroxymutase isogenes being responsible for this conversion. While the gene involved in enterobactin synthesis (entC) was known, the corresponding gene for menaquinone biosynthesis (menF) was not but has now been identified and sequenced. The amino acid sequence of MenF is 23.5% identical and 57.8% similar to that of EntC.

Microbial production of specifically ring-13C-labelled 4-hydroxybenzoic acid.

When transformed with a recombinant vector carrying the ubiC gene (encoding chorismate pyruvate-lyase, EC 4.1.3.27) the triple mutant (Phe-, Trp-, Tyr-) Klebsiella pneumoniae 62-1 excretes 4-hydroxybenzoic acid instead of chorismic acid. The recombinant strain can be used to produce in high yield specifically ring-labelled 4-hydroxybenzoic acid from isotopically labelled glucose.

Purification and characterization of the pyridoxol-5'-phosphate:oxygen oxidoreductase (deaminating) from Escherichia coli.

The E. coli gene pdxH encoding pyridoxol-5'-phosphate:oxygen oxidoreductase (deaminating) (EC 1.4.3.5, PdxH) was cloned, located to phage 20B5 of the library of Kohara et al. (Kohara, Y, Akiyama, K. and Isono K. (1987) Cell 50, 495-508) and assigned to a stretch between 36.0 and 36.1 min of the E. coli chromosome. The gene was overexpressed as a MBP/PdxH fusion protein. The fusion protein was purified by affinity chromatography on an amylose resin and hydrolyzed in the presence of protease 'factor Xa' resulting in homogeneous PdxH protein after another column chromatography. Both the MBP/PdxH fusion protein and the PdxH protein were characterized. Both enzymes are FMN-dependent enzymes which oxidize pyridoxol phosphate and pyridoxamine phosphate in the presence of oxygen to pyridoxal phosphate. Km values of both proteins were similar ranging from 350 to 400 microM for the two substrates. The enzymes did not accept non-phosphorylated substrates. Kinetic data indicate that the enzyme (MBP/PdxH) is product inhibited (Ki 8 microM) by pyridoxal phosphate as a mixed type inhibitor. As revealed by gel exclusion chromatography a minor fraction of the fusion protein formed a dimer, whereas the bulk amount of protein was a monomer. No indication was found that the PdxH protein forms a dimer. The monomer was shown to be catalytically active.

Microbial production of (+)-trans-isochorismic acid.

Two methods are described for the preparation of enantiomerically pure (+)-trans-isochorismic acid, an important metabolite of the postchorismate pathway. Both methods can be employed to prepare isotopically labeled isochorismic acid. One of the two methods is suitable to prepare bulk quantities of isochorismic acid using a recombinant strain of Klebsiella pneumoniae 62-1. (c) 1995 John Wiley & Sons, Inc.

Properties of isochorismate hydroxymutase from Flavobacterium K3-15.

Isochorismate hydroxymutase (isochorismate synthase, E.C. 5.4.99.6) catalyzes the interconversion of chorismic acid [1] and isochorismic acid [2]. The enzyme was extracted from a Flavobacterium K3-15 that overproduces vitamin K2 (i.e., menaquinones) and was purified to homogeneity. The N-terminal amino acid sequence and the mol wt (36,240 +/- 100 daltons) were determined by ms following SDS PAG electrophoresis. The enzyme was characterized (stability, cofactor requirement, isoelectrical point), and antibodies were raised which showed no cross reactivity with isochorismate hydroxymutase from Escherichia coli and Enterobacter aerogenes 62-1. The kinetic data of the enzyme are different from those observed for the corresponding enzyme from Escherichia coli and Galium mollugo.

Immobilization of isochorismate hydroxymutase. Comparison of native versus immobilized enzyme.

Partially purified isochorismate hydroxymutase (isochorismate synthase, E.C. 5.4.99.6) from Flavobacterium K3-15, a vitamin K overproducer, was immobilized on CNBr-activated Sepharose 4B, alkylamine glass substituted with glutardialdehyde, and aminohexyl Sepharose 4B substituted with glutardialdehyde. The immobilized enzyme exhibited a lower specific activity but a broader pH tolerance and a higher thermostability than the soluble enzyme. The stability of the enzyme was greatly increased by immobilization. Isochorismic acid, which is not commercially available, was prepared by a constant flow incubation.

Growth response to 4-hydroxy-L-threonine of Escherichia coli mutants blocked in vitamin B6 biosynthesis.

Mutants of Escherichia coli (pdx B and pdx C) which are blocked in the biosynthesis of pyridoxol (vitamin B6) showed a growth response to 4-hydroxy-L-threonine. This observation constitutes the first direct evidence in support of the view that 4-hydroxy-L-threonine is implicated in the biosynthesis of vitamin B6. 1-Aminopropan-2,3-diol, the decarboxylation product of 4-hydroxy-L-threonine, does not support the growth of these mutants. Deuterium from deuterium-labelled 1-aminopropan-2,3-diol was not incorporated into pyridoxol.