Lessons From the Studies of a CC Bond Forming Radical SAM Enzyme in Molybdenum Cofactor Biosynthesis

Abstract

MoaA is one of the founding members of the radical S-adenosyl-_L-methionine (SAM) superfamily, and together with the second enzyme, MoaC, catalyzes the construction of the pyranopterin backbone structure of the molybdenum cofactor (Moco). However, the exact functions of both MoaA and MoaC had remained ambiguous for more than 2 decades. Recently, their functions were finally elucidated through successful characterization of the MoaA product as 3',8-cyclo-7,8-dihydro-GTP (3',8-cH₂GTP), which was shown to be converted to cyclic pyranopterin monophosphate (cPMP) by MoaC. 3',8-cH₂GTP was produced in a small quantity and was highly oxygen sensitive, which explains why this compound had previously eluded characterization. This chapter describes the methodologies for the characterization of MoaA, MoaC, and 3',8-cH₂GTP, which together significantly altered the view of the mechanism of the pyranopterin backbone construction during the Moco biosynthesis. Through this chapter, we hope to share not only the protocols to study the first step of Moco biosynthesis but also the lessons we learned from the characterization of the chemically labile biosynthetic intermediate, which would be informative for the study of many other metabolic pathways and enzymes.

Keywords: Biosynthesis; Biosynthetic intermediate; Isolation; Molybdenum cofactor; Oxygen-sensitive small molecules; Pterin; Quantitative characterization; Radical SAM enzymes.