Recycling of Overactivated Acyls by a Type II Thioesterase during Calcimycin Biosynthesis in Streptomyces chartreusis NRRL 3882

Type II thioesterases typically function as editing enzymes removing acyl groups which have been mis-conjugated to acyl carrier proteins during polyketide secondary metabolite biosynthesis as a consequence of biosynthetic errors. Streptomyces chartreusis NRRL 3882 produces the pyrrole polyether ionophoric antibiotic and we have identified the presence of a putative type II thioesterase like sequence, calG, within the biosynthetic gene cluster involved in the antibiotic9s synthesis. However targeted gene mutagenesis experiments in which calG was inactivated in the organism did not lead to a decrease in calcimycin production but rather reduced the strain9s production of its main biosynthetic precursor, cezomycin. Results from in vitro activity assays of purified, recombinant CalG protein indicated that it was involved in the hydrolysis of cezomycin-CoA, as well as other acyl CoAs, but was not active toward 3-S-N-acetylcysteamine (SNAC - the mimic of the polyketide chain releasing precursor). Further investigation of the enzyme9s activity showed that it possessed a cezomycin-CoA hydrolysis K m of 0.67 mM and a k cat of 17.77 min -1 and was significantly inhibited by the presence of Mn 2+ , and Fe 2+ divalent cations. Interestingly when S. chartreusis NRRL 3882 was cultured in the presence of inorganic nitrite, NaNO 2, it was observed that the production of calcimycin rather than cezomycin was promoted. Also that the supplementation of S. chartreusis NRRL 3882 growth medium with the divalent cations Ca 2+ , Mg 2+ , Mn 2+ , and Fe 2+ had a similar effect. Taken together these observations suggest that CalG is not responsible for mega-synthase polyketide precursor chain release during the synthesis of calcimycin nor for retaining the catalytic efficiency of the mega-synthase enzyme complex as is supposed to be the function for type II thioesterases. Rather our results suggest that CalG is a dedicated thioesterase that prevents the accumulation of cezomycin-CoA when intracellular nitrogen is limited, an apparently new and previously unreported function of Type II thioesterases. Importance Type II thioesterases (TEIIs) are generally regarded as being responsible for removing aberrant acyl groups that block polyketide production thereby maintaining the efficiency of the mega-synthase involved in this class of secondary metabolite9s biosynthesis. Specifically this class of enzyme is believed to be involved in editing mis-primed precursors, controlling initial units, providing key intermediates and releasing final synthetic products in the biosynthesis of this class of secondary metabolite. Our results indicate that the putative TEII, CalG, present in the calcimycin (A23187) producing organism Streptomyces chartreusis NRRL 3882 is not important either for the retention of catalytic efficiency of, or the release of the product compound from, the mega-synthase involved in calcimycin biosynthesis. Rather the enzyme is involved in regulating/controlling the pool size of the calcimycin biosynthetic precursor, cezomycin, by hydrolysis of its CoA derivative. This novel function of CalG suggests a possible additional activity for enzymes belonging to the TEII protein family and promotes better understanding of the overall biosynthetic mechanisms involved in the production of this class of secondary metabolite.