TY - JOUR

T1 - Decrypting the programming of β-methylation in virginiamycin M biosynthesis

AU - Collin, Sabrina

AU - Cox, Russell J.

AU - Paris, Cédric

AU - Jacob, Christophe

AU - Chagot, Benjamin

AU - Weissman, Kira J.

AU - Gruez, Arnaud

N1 - Funding Information: We acknowledge financial support from the Agence Nationale de la Recherche (grant numbers ANR-11-JSV8-003-01, PKS-PPIs; ANR-16-CE92-0006-01, PKS STRUCTURE; and, ANR-20-CE93-0002-01, PKSOx to K.J.W.), the Université de Lorraine and the Centre National de la Recherche Scientifique (CNRS). We also acknowledge J. Davison for help with the molecular biology, Omar A. Rifi for assistance with protein production and modification, and W. Shepard and M. Savko (Soleil Synchrotron, Proxima2) as well as J. Perez and A. Thureau (Soleil Synchrotron, Swing) for help with data acquisition. Crystal screening for diffraction quality and acquisition of NMR data were carried out on the Plateforme de Biophysique et Biologie Structurale (B2S) (IBSLor, UMS2008, CNRS-UL-INSERM). Analytical chemistry was performed on the Structural and Metabolomics Analyses Platform (PASM), SF4242, Université de Lorraine, EFABA, Vandœuvre‐lès‐Nancy, France.

PY - 2023/3/10

Y1 - 2023/3/10

N2 - During biosynthesis by multi-modular trans-AT polyketide synthases, polyketide structural space can be expanded by conversion of initially-formed electrophilic β-ketones into β-alkyl groups. These multi-step transformations are catalysed by 3-hydroxy-3-methylgluratryl synthase cassettes of enzymes. While mechanistic aspects of these reactions have been delineated, little information is available concerning how the cassettes select the specific polyketide intermediate(s) to target. Here we use integrative structural biology to identify the basis for substrate choice in module 5 of the virginiamycin M trans-AT polyketide synthase. Additionally, we show in vitro that module 7, at minimum, is a potential additional site for β-methylation. Indeed, analysis by HPLC-MS coupled with isotopic labelling and pathway inactivation identifies a metabolite bearing a second β-methyl at the expected position. Collectively, our results demonstrate that several control mechanisms acting in concert underpin β-branching programming. Furthermore, variations in this control – whether natural or by design – open up avenues for diversifying polyketide structures towards high-value derivatives.

AB - During biosynthesis by multi-modular trans-AT polyketide synthases, polyketide structural space can be expanded by conversion of initially-formed electrophilic β-ketones into β-alkyl groups. These multi-step transformations are catalysed by 3-hydroxy-3-methylgluratryl synthase cassettes of enzymes. While mechanistic aspects of these reactions have been delineated, little information is available concerning how the cassettes select the specific polyketide intermediate(s) to target. Here we use integrative structural biology to identify the basis for substrate choice in module 5 of the virginiamycin M trans-AT polyketide synthase. Additionally, we show in vitro that module 7, at minimum, is a potential additional site for β-methylation. Indeed, analysis by HPLC-MS coupled with isotopic labelling and pathway inactivation identifies a metabolite bearing a second β-methyl at the expected position. Collectively, our results demonstrate that several control mechanisms acting in concert underpin β-branching programming. Furthermore, variations in this control – whether natural or by design – open up avenues for diversifying polyketide structures towards high-value derivatives.

UR - http://www.scopus.com/inward/record.url?scp=85149970867&partnerID=8YFLogxK

U2 - 10.1038/s41467-023-36974-3

DO - 10.1038/s41467-023-36974-3

M3 - Article

C2 - 36899003

AN - SCOPUS:85149970867

VL - 14

JO - Nature Communications

JF - Nature Communications

SN - 2041-1723

M1 - 1327

ER -