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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, Ian J., Deng, Z., 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). ISSN 0099-2240. (doi:10.1128/AEM.00587-18) (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided)

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Official URL
http://dx.doi.org/10.1128/AEM.00587-18

Abstract

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 Km of 0.67 mM and a kcat of 17.77 min-1 and was significantly inhibited by the presence of Mn2+ and Fe2+ divalent cations. Interestingly, when S. chartreusis NRRL 3882 was cultured in the presence of inorganic nitrite, NaNO2, 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 Ca2+, Mg2+, Mn2+, and Fe2+ 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
DOI/Identification number: 10.1128/AEM.00587-18
Uncontrolled keywords: Antibiotics; Bacteria; Biochemistry; Chains; Enzyme activity; Genes; Hydrolysis; Ketones; Metabolites; Positive ions; Recombinant proteins; Sodium compounds; Tissue, Acyl carrier proteins; Biosynthetic gene cluster; Biosynthetic precursors; Catalytic efficiencies; Divalent cation; Mutagenesis experiment; Secondary metabolites; Thioesterases, Biosynthesis, antibiotics; bacterium; bioassay; cation; enzyme; enzyme activity; gene; gene expression; growth; hydrolysis; protein; secondary metabolite, Streptomyces chartreusis
Subjects: Q Science > QR Microbiology
Divisions: Faculties > Sciences > School of Physical Sciences > Functional Materials Group
Depositing User: I. Bruce
Date Deposited: 01 Feb 2019 13:09 UTC
Last Modified: 05 Sep 2019 15:01 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/71926 (The current URI for this page, for reference purposes)
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