Pseudouridimycin Biosynthesis: Biochemical Characterization of the Glucose–Methanol–Choline (GMC) Family Oxidoreductase, PumI

Pseudouridimycin (PUM) is a <i>C</i>-nucleoside antibiotic that selectively inhibits bacterial RNA polymerase (RNAP) with remarkable potency. It binds to the nucleoside triphosphate (NTP) entry region in the RNAP active site by mimicking uridine-5'-triphosphate (UTP), thus blocking RNA synthesis in bacteria. Since PUM does not inhibit human RNAP, it presents a highly selective scaffold for clinical applications. Besides its unique mode of action, PUM's peptidyl <i>C</i>-nucleoside structure comprises a rare pseudouridine (PU) moiety linked to an <i>N</i>-hydroxylated dipeptide, which is crucial for binding interactions with RNAP. Recently, the biosynthetic gene cluster (BGC) and a putative pathway have been reported for PUM biosynthesis. However, the investigation of the biosynthetic enzymes is still in its infancy. Here, we report detailed biochemical characterization of a flavin-dependent glucose-methanol-choline (GMC) family oxidoreductase, PumI, from <i>Streptomyces rimosus</i> (SrPumI) through substrate scope, computational modeling, mutational, kinetic, and mechanistic studies. Our studies have indicated that PumI preferentially accepts the native <i>C</i>-nucleoside substrate (PU) over <i>N</i>-nucleosides and acts as a gatekeeper in PUM biosynthesis. Our mutational analysis identified two active site histidines (His454 and His455) and two asparagines (Asn90 and Asn499) in SrPumI as potential flavin-binding residues. We propose His455 as the critical base for initiating catalysis based on our biochemical experiments and bioinformatics analysis. Additionally, Gln297 and Met58 were found to be important for substrate (PU) coordination. Based on these experiments, a mechanism has been proposed for PumI. We believe this work will provide new insights into PUM biosynthesis, enabling pathway engineering to prepare novel PUM derivatives for prospective therapeutic applications.