In this paper, we described detailed protocols of expression, purification, and characterization of MoaA and MoaC, as well as the isolation and characterization of 3′,8-cH2GTP. The in situ 13C NMR assay method was also described so that anyone interested can determine the product of MoaA without going through extensive purification in anaerobic glove box. We hope these protocols are informative to whoever interested in Moco biosynthesis or any other related systems.
This review summarizes the functions and mechanisms of the emerging group of radical SAM enzymes that catalyze C-C bond formations during natural products and cofactor biosynthesis. The review focuses on the roles of these enzymes in the biosynthetic pathways, and the key mechanistic questions relevant to many of these enzymes. Numbers of C-C bond forming radical SAM enzymes are increasing, but their mechanistic characterizations are still in its infancy.
A biochemistry perspective summarizing our studies on the Moco and antifungal peptidyl nucleoside biosyntheses came out as ASAP. This perspective summarizes the works mainly achieved by Brad Hover, Ph.D., and Edward Lilla (Ph.D. to be soon…!). These discoveries were made through characterization of unique C-C bond forming radical SAM enzymes. Congratulations to the two talented students!!
Our recent discovery of novel radical SAM enzyme that catalyzes free-radical mediated C-C bond formation during nikkomycin and polyoxin biosynthesis was published online in Nature Chemical Biology. The work is highlighted on the journal’s top page. Our studies showed that biosynthetic routes to nikkomycins and polyoxins proceeds via a bicyclic intermediate, octosyl acid phosphate (OAP), formed by a free radical mediated ring closure catalyzed by the radical SAM enzyme, PolH. We also propose OAP to be a common biosynthetic intermediate for various antifungal nucleosides, and PolH as the genetic marker for genome mining discovery of these natural products. Edward A. Lilla, a graduate student, performed all the characterizations described in the paper.
Our latest publication about pneumocandin biosynthesis in collaboration with Dr. Gerald Bills at the University of Texas was accepted to ACS Chemical Biology. This work revealed the mechanism of product release from lipid synthesizing PKS, and its application to create pneumocandin analogs. Anna performed 1,3-glucan synthase inhibition assays of the structurally novel pneumocandin analogs.
This paper describes successful entrapment of previously uncharacterized reaction intermediate of MoaC reaction using an uncleavable substrate analog, 3′,8-cH2GTP. Intriguingly, the trapped intermediate was tightly bound to MoaC, likely through covalent modification, making the analog as the first mechanism-based inhibitor of bacterial Moco biosynthesis. The results revealed that the unique tetracyclic structure of cPMP (MoaC product) was constructed via a concerted formation of pterin and cyclic phosphate rings, which is distinct from previously proposed stepwise mechanism. Brad found the irreversible inhibition and performed most of the basic characterization, and Edward performed some nice follow up experiments including the MS characterization of the inhibited MoaC.