Purification, characterization, cDNA cloning and expression of a novel ketoreductase from Zygosaccharomyces rouxii.

A novel ketoreductase isolated from Zygosaccharomyces rouxii catalyzes the asymmetric reduction of selected ketone substrates of commercial importance. The 37.8-kDa ketoreductase was purified more than 300-fold to > 95% homogeneity from whole cells with a 30% activity yield. The ketoreductase functions as a monomer with an apparent Km for 3,4-methylenedioxyphenyl acetone of 2.9 mM and a Km for NADPH of 23.5 microM. The enzyme is able to effectively reduce alpha-ketolactones, alpha-ketolactams, and diketones. Inhibition is observed in the presence of diethyl pyrocarbonate, suggesting that a histidine is crucial for catalysis. The 1.0-kb ketoreductase gene was cloned and sequenced from a Z. rouxii cDNA library using a degenerate primer to the N-terminal sequence of the purified protein. Furthermore, it was expressed in both Escherichia coli and Pichia pastoris and shown to be active. Substrate specificity, lack of a catalytic metal, and extent of protein sequence identity to known reductases suggests that the enzyme falls into the carbonyl reductase enzyme class.

Enantioselective reduction of 3,4-methylene-dioxyphenylacetone using Candida famata and Zygosaccharomyces rouxii.

In an effort to prepare 3,4-methylene-dioxyphenyl-(S)-isopropanol from 3,4-methylene-dioxyphenylacetone, an initial screen of microbes indicated that Candida famata could catalyze this reaction efficiently at low substrate concentration. A dilute, large-scale process was developed to provide experimental material for the chemical synthesis to be explored. However, the productivity number of this process [0.134 g product (g wet weight cells)-1 day-1 was too low to be practical. C. famata was also extremely sensitive to concentrations of both the ketone and the alcohol greater than 2 g/l. A more extensive screen of yeast and fungi revealed that Zygosaccharomyces rouxii was more tolerant to higher substrate concentrations and had a higher productivity number [0.8 g (g wet weight cells)-1 day-1]. These characteristics suggested that Z. rouxii could be used in a large-scale process at high substrate concentrations.

Biosynthetic studies on antibiotic A47934.

A47934, a peptide antibiotic produced by Streptomyces toyocaensis, belongs to the glycopeptide class of compounds which includes ristocetin and vancomycin. Incorporation studies with radioisotope-labeled substrates indicated that tyrosine, p-hydroxyphenylglycine, p-hydroxyphenylglyoxylate, acetate, and sulfate were efficiently incorporated into A47934. This is consistent with the reported biosynthesis of other glycopeptide antibiotics. Prototrophic mutants blocked in antibiotic biosynthesis were isolated at a low frequency (0.4%) after mutagenesis. Secretor-convertor pairings of the 36 mutants obtained demonstrated that they belonged to three classes: two groups of secretor-convertor pairs and a larger group of mutants that did not make antibiotic under any condition tested. Neither the secretor-convertor studies not supplementation of the cultures with putative biosynthetic intermediates was useful in identifying the location of the biosynthetic blocks. All studies to determine the timing of the sulfate addition step in the biosynthesis indicated that the sulfate is added prior to the formation of intermediates that possess antimicrobial activity.

The in vitro interaction of naphthyridinomycin with deoxyribonucleic acids.

The binding of naphthyridinomycin (NAP) to deoxyribonucleic acid was investigated using radioisotope labeled antibiotic. Dithiothreitol (DTT) enhances complex formation in a concentration dependent fashion but was found to be slightly inhibitory at concentrations above 10 mM. [C3H3]-NAP-DNA complexes, formed in the presence or absence of reducing reagents, were stable to Sephadex G-25 chromatography and precipitation with ethanol, indicating a strong bond formed between the drug and DNA. Time course studies showed that the difference between the binding of activated and non-activated antibiotic was a DTT-dependent burst. This was followed by a second phase of binding which was similar in both the activated and non-activated antibiotics. The activation of the antibiotic by DTT was a reversible reaction at pH 7.9. The activated form at pH 5.0 was extremely stable and did not revert to the unactivated form even after an 8-h incubation period. Antibiotic-DNA complex formation was pH independent between pH 5.0 and 7.0 for activated NAP. The non-activated antibiotic bound to DNA much better at pH 5.0 than at physiological pH values. Release of antibiotic from complexes (as followed by long term dialysis) formed in the presence of DTT and at pH 5.0 was biphasic, suggesting that the drug can bind to DNA in more than one way. A constant rate of antibiotic release was observed at pH 7.9 with or without DTT. At pH 2.0 and pH 12.0, greater than 95% of the antibiotic is released from the complexes. Most of the acid released antibiotic is NAP while most of the base released antibiotic had decomposed to a more polar compound. NAP binds well to calf thymus DNA, poly(dG) . poly(dC), and T4 DNA but shows significantly less affinity for poly(dA) . poly(dT), poly(dA . dT) . poly(dA . dT), poly(dG), poly(dC), poly(dI) . poly(dC) or poly(dG . dC) . poly(dG . dC). This specificity of NAP for DNA is similar to that observed for the pyrrolo(1,4)benzodiazepine antibiotics and saframycin A and S; all of which bind to double stranded DNA through their carbinolamine or masked carbinolamine functionalities. Two mechanisms which can explain the need for activation of NAP are also proposed.

The interaction of cyanonaphthyridinomycin with DNA.

The characteristics of the in vitro interaction of cyanonaphthyridinomycin (CYANO) with DNA are described. Unlike naphthyridinomycin (NAP), CYANO is extremely dependent on reductive activation with dithiothreitol (DTT) to bind DNA. The reaction of CYANO with DNA is kinetically slower than that observed for NAP and is still linear after six hours incubation at room temperature. The extent of binding is pH dependent with acidic pH being inhibitory. CYANO, as with NAP, appears to bind to dG:dC base pairs in the minor groove of double stranded DNA. Studies using [C(3)H3:(14)CN] CYANO demonstrated that the cyanide group is lost when the drug binds to DNA. In the absence of DNA but in the presence of DTT, cyanide is still released from CYANO and the extent of release is also inhibited by acid pH conditions. These results suggest that the cyanide group comes off prior to binding of the antibiotic to DNA. The rate limiting step in the reaction of CYANO with DNA would appear to be the release of cyanide from the drug molecule.

Naphthyridinomycin, a DNA-reactive antibiotic.

Naphthyridinomycin is a novel quinone antibiotic that is produced in liquid shake cultures by Streptomyces lusitanus. Fermentation studies have shown that this antibiotic is produced maximally after 96 h of cell growth. L-[methyl-3H]methionine efficiently labels naphthyridinomycin when it is added to a fermentation mixture 24 h before culture is harvested. Unlabeled and radioactively labeled naphthyridinomycin were used to determine the mechanism of action of this unique antibiotic. Naphthyridinomycin inhibited bacterial growth primarily by inhibiting DNA synthesis. The structural similarity between naphthyridinomycin and the saframycins suggested that naphthyridinomycin might inhibit DNA synthesis by binding to the template. In vitro studies with radiolabeled naphthyridinomycin indicated that this antibiotic does specifically bind to calf thymus DNA. The binding reaction was enhanced by adding sulfhydryl-containing compounds; dithiothreitol was the best activating agent. DNA-naphthyridinomycin complexes were a poor substrate for enzymes that catalyze DNA-directed DNA and RNA syntheses. These results showed that naphthyridinomycin is similar to the saframycins in its reactivity toward DNA and suggested that the mechanism by which naphthyridinomycin inhibits DNA synthesis is through its ability to bind specifically to the DNA template of the cell.

Biosynthesis of prostaglandin D2, 15-ketoprostaglandin E2, and hydroxy fatty acids by ram seminal vesicle microsomes.

At high arachidonic acid concentrations (164 micrometer) and without exogenous cofactors, ram seminal vesicle microsomes produced prostaglandin E2 and two less polar products, identified as prostaglandin D2 and 15-ketoprostaglandin E2. The ratio of the biosynthetic products formed depended on the exogenous cofactor and on the arachidonic acid concentration. At high arachidonic acid concentrations (greater than 150 micrometer), tryptophan, phenol, and glutathione stimulated prostaglandin E2 formation, but each affected the formation of the other prostaglandins differently. Ascorbic acid markedly stimulated hydroxy fatty acid formation. GLC-mass spectral analysis of the hydroxy fatty acid fraction indicated the presence of 11-hydroxy-5,8,12,14-eicosatetraenoic acid, 15-hydroxy-5,8,11,13-eicosatetraenoic acid, and 12-hydroxy-5,8,10-heptadecatrienoic acid. At low arachidonic acid concentrations (30 micrometer), glutathione still stimulated prostaglandin E2 biosynthesis, but the other cofactors stimulated 6-ketoprostaglandin F1a and hydroxy fatty acid formation.

Dual effects of glucose on dicarboxylic acid transport in Kluyveromyces lactis.

The properties of succinate uptake in succinate-grown Kluyveromyces cells were examined. The rate of succinate transport at 15C exhibits an approximate V-max of 1.2 mumol times h-1 times mg-1 dry weight of cells and an apparent K-m of 18 muM. The uptake process appears to be tightly coupled to metabolism. L-Malate, fumarate, and alpha-ketoglutarate were the only other dicarboxylates tested, which were found to inhibit succinate transport. The aggreement between the order of inhibition of succinate transport by these dicarboxylates and their rates of uptake, as well as the competitive nature of the inhibition are all consistent with the existence of a common carrier system showing specificity for dicarboxylates of the TCA cycle. Cells transferred from succinate to glucose medium rapidly lose their ability to transport succinate. Glucose-grown cells also exhibit an inability to oxidize dicarboxylates or to use them for growth without a very long lag. The dicarboxylate uptake system, therefore, appears to be subject to a strong catabolite repression. The depression of the succinate transport system requires the presence of succinate, as well as low concentrations of glucose.