Binding of NADP+ triggers an open-to-closed transition in a mycobacterial FabG β-ketoacyl-ACP reductase

The ketoacyl-acyl carrier protein (ACP) reductase FabG catalyzes the NADPH/ NADH dependent reduction of a-ketoacyl-ACP substrates to beta-hydroxyacyl-ACP products, the first reductive step in the fatty acid biosynthesis elongation cycle. FabG proteins are ubiquitous in bacteria and are part of the type II fatty acid synthase system. Mining the Mycobacterium smegmatis genome uncovered several putative FabG-like proteins. Among them, we identified M. smegmatis MSMEG_ 6753 whose gene was found adjacent to MSMEG_ 6754, encoding a recently characterized enoyl-CoA dehydratase, and to MSMEG_ 6755, encoding another potential reductase. Recombinantly expressed and purified MSMEG_ 6753 exhibits ketoacyl reductase activity in the presence of acetoacetyl- CoA and NADPH. This activity was subsequently confirmed by functional complementation studies in a fabG thermosensitive Escherichia coli mutant. Furthermore, comparison of the apo and the NADP(+)-bound MSMEG_6753 crystal structures showed that cofactor binding induces a closed conformation of the protein. A.MSMEG_ 6753 deletion mutant could be generated in M. smegmatis, indicating that this gene is dispensable for mycobacterial growth. Overall, these results showcase the diversity of FabG-like proteins in mycobacteria and new structural features regarding the catalytic mechanism of this important family of enzymes that may be of importance for the rational design of specific FabG inhibitors.