Kimberlee K. Wallace

Ammonium effects on streptonigrin biosynthesis by Streptomyces flocculus

SummaryA defined medium containing glucose and ammonium as the sole carbon and nitrogen sources was developed to support growth and streptonigrin production. In this defined medium, increased initial levels of ammonium resulted in increased growth suggesting that nitrogen is the growth limiting nutrient. In some cases, increased initial ammonium levels resulted in decreased specific streptonigrin productivity, suggesting that nitrogen regulatory mechanisms may adversely affect streptonigrin biosynthesis. This suggestion that nitrogen regulation adversely affects antibiotic biosynthesis is further supported by results from two studies in which the ammonium supply to the cells was controlled. In the first study, streptonigrin productivity and final titer were enhanced by the addition of an ammonium trapping agent. In the second experiment, when ammonium chloride was fed slowly throughout the course of cultivation, the production phase was lengthened and the maximum antibiotic concentration was enhanced compared to the batch controls containing either the same initial or the same total ammonium chloride levels. Although our results indicate streptonigrin production may be subject to nitrogen regulatory mechanisms, the effect of nitrogen on streptonigrin production cannot be strictly correlated to the extracellular ammonium concentration. In fact, we observed that when ammonium was depleted from the medium, streptonigrin production ceased.

Kinetic and stereoelectronic effects of a fluorine substituent on the reaction catalysed by an NADPH-dependent cyclohex-1-enylcarbonyl CoA reductase

The introduction of a fluorine atom at C-3 of cyclohex-1-enecarbonyl CoA has a dramatic effect on the processing of the fluorinated over the natural analogue by NADPH dependant cyclohex-1-enecarbonyl CoA reductase from Streptomyces collinus; the fluorinated analogue is processed with a five fold increase in Vmax and kinetic isotope studies suggest that hydride delivery is only partially rate limiting in the latter case; the enzyme also shows a small kinetic preference for axial over equatorial fluorine at C-3 of 3-fluorocyclohex-1-encarbonyl CoA 4.

Methylation Pathways in Antibiotic Producing Streptomycetes

Most secondary metabolites produced in plants and microorganisms contain methyl groups donated from S-adenosylmethionine. While many of these are transferred to oxygen and nitrogen atoms, others involve C-methylation to form carbon-carbon bonds. Examples of pathways in which such a reaction occurs include the antibiotics thiostrepton1, indolmycin2, actinomycin3, thienamycin4, anthramycin5, and streptonigrin6. The methyltransferases which catalyze some of these reactions have been identified in cell-free extracts and, in some instances, purified to homogeneity. Table 1 lists properties of several O- and N-methyl transferases which have been characterized from plant and microbial sources and Table 2 summarizes the properties of several C-methy1trans ferases, including the tryptophan C-methy1transferase which is the topic of this paper. It should be noted that in all cases which have been studied7,8, the mechanism is of the ordered bi-bi type with S-adenosylmethionine binding first and its product, S-adenosylhomocysteine, being released last. Also, in most cases that have been studied, the methyl group is transferred with inversion of configuration, indicating a direct transfer12, but in at least two cases, those of the antibiotics thienamycin and thiostrepton, retention of configuration is observed, suggesting a two—step methylation process1,4. Finally, most enzymes are inhibited by S-adenosylhomocysteine, although the KiS are often above the Km for S-adenosylmethionine, which is interesting in light of methyltransferases observed in many other systems which have been found to have greater affinity for the inhibitor than for the substrate13. However, in crude systems the amount of S-adenosylhomocysteine present may be overestimated unless the S-adenosylhomocysteine metabolizing enzymes are specifically inhibited during the determination of the Ki.