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Lohman, J. R.

Publications and source records attributed to Lohman, J. R..

2 recordsLinked to original sources

Active E. coli heteromeric acetyl-CoA carboxylase forms polymorphic helical tubular filaments

The Escherichia coli heteromeric acetyl-CoA carboxylase (ACC) has three functional subunits assumed to form an elusive catalytic complex and they are involved in allosteric and transcriptional regulation. The E. coli ACC represents almost all ACCs from pathogenic bacteria, making it a key antibiotic development target to fight growing antibiotic resistance. Furthermore, it is a model for cyanobacterial and plant plastid ACCs as biofuel engineering targets. Here, we report the catalytic E. coli ACC complex surprisingly forms tubes rather than dispersed particles. The cryo-EM structure reveals key protein-protein interactions underpinning efficient catalysis and how transcriptional regulatory roles are masked during catalysis. Discovering the protein-protein interaction interfaces that facilitate catalysis, allosteric, and transcriptional regulation provides new routes to engineering catalytic activity and new targets for drug discovery. One-Sentence SummaryBacterial heteromeric acetyl-CoA carboxylase forms tubes to promote efficient catalysis and mask transcriptional regulation.

biochemistry↗

Structures of chloramphenicol acetyltransferase III and E. coli β-ketoacylsynthase III co-crystallized with partially hydrolysed acetyl-oxa(dethia)CoA

Acetyl-CoA is a reactive metabolite that non-productively hydrolyzes in a number of enzyme active sites on the crystallization time frame. In order to elucidate enzyme:acetyl-CoA interactions leading to catalysis, acetyl-CoA substrate analogs are needed. One possible analog for use in structural studies is acetyl-oxa(dethia)CoA (AcOCoA), where the thioester sulfur of CoA is replaced by an oxygen. Here we present structures of chloramphenicol acetyltransferase III (CATIII) and E. coli ketoacylsynthase III (FabH) from crystals grown in the presence of partially hydrolyzed AcOCoA and the respective nucleophile. Based on the structures, the behaviour of AcOCoA differs between the enzymes, with FabH reacting with AcOCoA and CATIII being unreactive. The structure of CATIII reveals insight into the catalytic mechanism, with one active site of the trimer having relatively clear electron density for AcOCoA and chloramphenicol, and the other active sites having weaker density for AcOCoA. One FabH structure has a hydrolyzed AcOCoA product oxa(dethia)CoA (OCoA) and the other FabH structure has an acyl-enzyme intermediate with OCoA. Together these structures provide preliminary insight into the use of AcOCoA for enzyme structure-function studies with different nucleophiles. SynopsisStable analogs of acetyl-CoA are needed to support structure-function studies of acetyltransferase enzymes. We report structures of two enzymes in the presence of an acetyl-CoA analog where the thioester is replaced by an ester.

biochemistry↗