Structural and mutational analysis of Methanosarcina mazei prenylated FMN synthase reveals the basis of its unique prenyl donor substrate specificity.

Abstract

Prenylated flavin mononucleotide (prFMN) is a flavin coenzyme that helps UbiD-family microbial enzymes catalyze the decarboxylation of α,β-unsaturated carboxylic acids. Since the UbiD-family decarboxylases are involved in important metabolic processes, such as the anaerobic catabolism of aromatic acids, bacterial ubiquinone biosynthesis, and the archaeal modified mevalonate pathway, prFMN is essential for many microorganisms. Biosynthesis of prFMN via the transfer of a dimethylallyl group to the N5 of reduced FMN and subsequent cyclization is catalyzed by a UbiX-family prenyltransferase called prFMN synthase (PFS). PFSs are unique because some accept dimethylallyl phosphate (DMAP) as the prenyl donor substrate instead of dimethylallyl diphosphate (DMAPP), which is a common donor substrate for many prenyltransferases. Structural and mutagenic analyses of PFS from the methanogenic archaeon Methanosarcina mazei were performed in the present study to elucidate the detailed mechanism underlying the unique donor substrate preference of PFSs. M. mazei PFS is DMAP-specific, but it can also accept DMAPP. The crystal structures of the enzyme in complex with FMN, both FMN and DMAP, or prFMN were solved, revealing the substrate-binding residues. Point mutations at a non-conserved residue, Thr163, near the substrate-binding site changed the donor substrate specificity, primarily affecting the catalytic rate rather than substrate recognition. The T163F mutant significantly decreased its activity toward DMAPP, becoming more specific to DMAP, whereas the T163Q mutant was completely inactive when DMAPP was used for the reaction. This study provides a deeper understanding of how PFS recognizes its substrates and synthesizes prFMN, emphasizing the importance of DMAP, an overlooked metabolite.