ABSTRACT
In an effort to uncover the structural motifs and biosynthetic logic of the relatively uncharacterized trans-acyltransferase polyketide synthases, we have begun the dissection of the enigmatic dehydrating bimodules common in these enzymatic assembly lines. We report the 1.98 Å resolution structure of a ketoreductase (KR) from the first half of a type A dehydrating bimodule and the 2.22 Å resolution structure of a dehydratase (DH) from the second half of a type B dehydrating bimodule. The KR, from the third module of the bacillaene synthase, and the DH, from the tenth module of the difficidin synthase, possess features not observed in structurally characterized homologs. The DH architecture provides clues for how it catalyzes a unique double dehydration. Correlations between the chemistries proposed for dehydrating bimodules and bioinformatic analysis indicate that type A dehydrating bimodules generally produce an α/ß-cis alkene moiety, while type B dehydrating bimodules generally produce an α/ß-trans, γ/δ-cis diene moiety.
Subject(s)
Acyltransferases/chemistry , Alcohol Oxidoreductases/chemistry , Bacterial Proteins/chemistry , Polyketide Synthases/chemistry , Acyltransferases/metabolism , Alcohol Oxidoreductases/metabolism , Bacillus/enzymology , Bacterial Proteins/metabolism , Polyketide Synthases/metabolismSubject(s)
Bacillus amyloliquefaciens/enzymology , Cysteamine/analogs & derivatives , Cysteamine/metabolism , Methyltransferases/metabolism , Polyketide Synthases/metabolism , Pseudomonas fluorescens/enzymology , Bacillus amyloliquefaciens/genetics , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Kinetics , Methyltransferases/genetics , Polyketide Synthases/genetics , Pseudomonas fluorescens/genetics , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
C-methyltransferases (MTs) from modular polyketide synthase assembly lines are relatively rare and unexplored domains that are responsible for installing α-methyl groups into nascent polyketide backbones. The stage at which these synthase-embedded enzymes operate during polyketide biosynthesis has yet to be conclusively demonstrated. In this work we establish the activity and substrate preference for six MTs from the gephyronic acid polyketide synthase and demonstrate their ability to methylate both N-acetylcysteamine- and acyl carrier protein-linked ß-ketoacylthioester substrates but not malonyl thioester equivalents. These data strongly indicate that MT-catalyzed methylation occurs immediately downstream of ketosynthase-mediated condensation during polyketide assembly. This work represents the first successful report of MT-catalyzed mono- and dimethylation of simple thioester substrates and provides the groundwork for future mechanistic and engineering studies on this important but poorly understood enzymatic domain.
