摘要
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 kcat/Km 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.
基金
supported by the National Key Research and Development Program of China(2023YFA0914500)
the National Science Foundation of China(32271487)
the National First-class Discipline Program of Light Industry Technology and Engineering(LITE2018-12)
the Program of Introducing Talents of Discipline to Universities(111-2-06).
