Studies of enzymes involved in the primary and secondary metabolism of antibiotic producing streptomyces

Priestley, Nigel David (1990) Studies of enzymes involved in the primary and secondary metabolism of antibiotic producing streptomyces. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

An NAD⁺ dependent L-valine dehydrogenase was purified 180 fold from Streptomyces cinnamonensis. The enzyme has a mass of 88 kDa and appears to be composed of two subunits of equal mass. The enzyme requires NAD⁺ as the only cofactor which cannot be replaced by NADP⁺. The enzymic activity is abolished by p-chloromercurybenzoate, Hg(I) ions, and Hg(II) ions. Neither uridine, thymidine, adenosine, guanosine, cytidine nor their mono- di- or triphosphates significantly affected the in vitro activity of the enzyme. The reductive amination reaction proceeds through an ordered ternary-binary mechanism with NADH being the first substrate to bind to the enzyme followed sequentially by 2-oxoisovalerate and ammonia. L-valine is the first product of the reductive deamination to dissociate from the enzyme complex followed by NAD⁺. The K_m values for L-valine and NAD⁺ are 1.3 ± 0.4 and 0.13 ± 0.09 mM respectively. The apparent K_m values for NADH, 2-oxoisovalerate, and ammonia are 0.074 ± 0.007, 0.81 ±0.08 and 55 ± 3 mM respectively. The pro-S hydrogen at the C-4' position of NADH is transferred to the substrate in the reductive amination reaction. It is proposed that this enzyme catalyses the first step in the oxidative catabolism of L-valine in this organism. The purification and characterisation of the enzyme responsible for catalysing the novel isobutyrate to n-butyrate rearrangement in S. cinnamonensis have been attempted. Improvements have been made to the g.c. assay developed by Reynolds (Ph.D. Thesis, University of Southampton, 1987) which for the first time allow the determination of accurate initial rates for the reaction in vitro and hence the specific activities of the enzyme. The activity of the enzyme in the crude extracts of S. cinnamonensis is of the order of 5 to 10 mUmg^-1. The K_m values for coenzyme-B_12 and n-butyryl-CoA were estimated as 4.5 ± 0.1 and 160 ± 10 μmoldm^-3 respectively. The activity of methylmalonyl-CoA mutase in the cell free system was determined by a novel u.v. assay which monitored the production of succinyl-CoA. The isobutyryl-CoA mutase is inhibited by CoASH but not by methylmalonyl-CoA or succinyl-CoA. The mutase enzyme was found to be inactivated by column chromatography although it was stable in the cell free system (half-life of 35.5 h at pH 7). The stability of the enzyme is improved by extensive dialysis suggesting that the loss of activity during chromatography is not due to the loss of any essential ion or cofactor. The rate of the reaction is reduced at high ionic strength although this has no effect on the absolute stability of the enzyme. It is thought that the inactivation is due to the dissociation of the native enzyme and subequent separation of its subunits. Attempts have been made to detect an enzyme activity in S. griseus ETH A7796 which can stereospecifically link the four units of nonactic acid to form the macrotetrolide nonactin. [8-³H]Nonactin acid, of high specific activity, has been synthesised and activated as its N-caprylcysteamine thioester and then used in feeding studies and an in vitro assay. When administered to a producing culture an incorporation of 42% of the label into nonactin was detected. No significant incorporation, however, could be detected in in vitro assays with broken cell systems.