Recycling of overactivated acyls by a type II thioesterase during calcimycin biosynthesis in Streptomyces chartreusis NRRL 3882.

Wu, H., Liang, J.., Gou, L., Wu, Q., Liang, W.-J., Zhou, X., Bruce, I.J., Deng, Z. and Wang, Z., 2018. Recycling of overactivated acyls by a type II thioesterase during calcimycin biosynthesis in Streptomyces chartreusis NRRL 3882. Applied and Environmental Microbiology, 84 (12), e00587-18.

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DOI: 10.1128/AEM.00587-18

Abstract

© 2018 American Society for Microbiology. Type II thioesterases typically function as editing enzymes, removing acyl groups that have been misconjugated 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 antibiotic's 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 strain's production of its biosynthetic precursor, cezomycin. Results from in vitro activity assays of purified, recombinant CalG protein indicated that it was involved in the hydrolysis of cezomycin coenzyme A (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 enzyme's 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, 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 megasynthase polyketide precursor chain release during the synthesis of calcimycin or for retaining the catalytic efficiency of the megasynthase 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.

Item Type:Article
ISSN:0099-2240
Group:Faculty of Science & Technology
ID Code:30858
Deposited By: Unnamed user with email symplectic@symplectic
Deposited On:13 Jun 2018 15:29
Last Modified:13 Jun 2018 15:29

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