Biochemical and Structural Insights into the Aminotransferase CrmG in Caerulomycin Biosynthesis

Caerulomycin A (CRM A <b>1</b>) belongs to a family of natural products containing a 2,2′-bipyridyl ring core structure and is currently under development as a potent novel immunosuppressive agent. Herein, we report the functional characterization, kinetic analysis, substrate specificity, and structure insights of an aminotransferase CrmG in <b>1</b> biosynthesis. The aminotransferase CrmG was confirmed to catalyze a key transamination reaction to convert an aldehyde group to an amino group in the <b>1</b> biosynthetic pathway, preferring l-glutamate and l-glutamine as the amino donor substrates. The crystal structures of CrmG in complex with the cofactor 5′-pyridoxal phosphate (PLP) or 5′-pyridoxamine phosphate (PMP) or the acceptor substrate were determined to adopt a canonical fold-type I of PLP-dependent enzymes with a unique small additional domain. The structure guided site-directed mutagenesis identified key amino acid residues for substrate binding and catalytic activities, thus providing insights into the transamination mechanism of CrmG