Summary of main observation and conclusion Chlorothricin (CHL),an archetypal member of the family of spirotetronate antibiotics,possesses a tetronate-containing pentacyclic aglycone that is conjugated with a modified ...Summary of main observation and conclusion Chlorothricin (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)-Iike 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-methyI-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.展开更多
文摘Summary of main observation and conclusion Chlorothricin (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)-Iike 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-methyI-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.
