Insights into the Functionalization of the Methylsalicyclic Moiety during the Biosynthesis of Chlorothricin by Comparative Kinetic Assays of the Activities of Two KAS III‐like Acyltransferases

Abstract

Summary of main observation and conclusionChlorothricin (CHL), an archetypal member of the family of spirotetronate antibiotics, possesses a tetronate‐containing pentacyclic aglycone that is conjugated with a modified methylsalicyclic acid (MSA) moiety through a disaccharide linkage. MSA is a polyketide product assembled by the iterative type I polyketide synthase ChlB1. Incorporation of this pharmaceutically important moiety into CHL relies on the activities of two distinct β‐Ketoacyl‐ACP synthase III (KAS III)‐like acyltransferases, ChlB3 and ChlB6, which function together to coordinate the transfer of MSA through ChlB2, a discrete acyl carrier protein (ACP). During the maturation of CHL, MSA needs to be further functionalized by C2‐O‐methylation and C5‐chlorination; however, timing of this functionalization process remains poorly understood. In this study, we report comparative kinetic assays of the activities of the two KAS III‐like acyltransferases ChlB3 and ChlB6 using substrates that vary in substitution extent and ACP carrier. ChlB3 prefers to transfer the immediately assembled 6‐methyl‐MSA moiety from ChlB1‐ACP to the discrete ACP ChlB2, from which this moiety is preferred to be transferred directly onto the molecule desmethylsalicyl‐CHL prior to C2‐O‐methylation and C5‐chlorination. Consequently, MSA functionalization appears to occur at the molecule level rather than at the covalently tethered protein level, i.e., ChlB1‐ACP or ChlB2. Both ChlB3 and ChlB6 are flexible in substrate tolerance, holding promise for CHL engineering‐based structural diversity by using variable MSA moiety.