Functional characterization of SgnI: a type II thioesterase critical for natamycin biosynthesis in Streptomyces gilvosporeus

Abstract

Two types of thioesterases are commonly found in natural product biosynthetic clusters: type I thioesterases, which release the final product from the biosynthetic complex, and type II thioesterases, which ensure biosynthetic fidelity by editing aberrant acyl carrier protein intermediates. In this study, we analyzed the structure and kinetic feature of SgnI, a type II thioesterase from the modular polyketide synthase natamycin biosynthetic cluster. Steady-state kinetic results revealed that SgnI preferentially hydrolyzes malonyl-CoA, with k_cat/K_m values that are 17.7-fold, 5.08-fold, and 1.30-fold higher compared to those for ethylmalonyl-CoA, acetyl-CoA, and methylmalonyl-CoA, respectively. This confirms that SgnI functions as an editing thioesterase. Furthermore, SgnI was shown to hydrolyze malonyl units from the phosphopantetheine arm of various acyl carrier domains. Structural modeling of SgnI revealed a wedge-shaped hydrophobic substrate-binding cleft, which restricts substrate size. To elucidate the molecular mechanisms underlying SgnI’s substrate specificity, molecular dynamics simulations were conducted on the SgnI-malonyl-CoA and SgnI-ethylmalonyl-CoA complexes. The smaller active site pocket of the SgnI-malonyl-CoA complex, coupled with enhanced interactions between active site residues and malonyl-CoA, likely contributes to its higher catalytic efficiency in hydrolyzing malonyl-CoA. These findings advance our understanding of thioesterase specificity and pave the way for engineering trans-acting thioesterases for use in biosynthetic assembly lines.

Keywords

Thioesterase / Catalytic mechanism / Molecular dynamics simulations / Biological Sciences / Biochemistry and Cell Biology

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Funding

National Key Research and Development Program of China(2023YFA0914500); National Science Foundation of China(32271487); National First-class Discipline Program of Light Industry Technology and Engineering(LITE2018-12); Program of Introducing Talents of Discipline to Universities(111-2-06)