Lessons From the Studies of a C C 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 bio-synthetic intermediate, which would be informative for the study of many other metabolic pathways and enzymes.

The First Step of Neurospora crassa Molybdenum Cofactor Biosynthesis: Regulatory Aspects under N-Derepressing and Nitrate-Inducing Conditions

“…In our work, we identified the amounts of the non-spliced NIT-7A encoding nit-7 gene transcript ( Figure 8D) to be likewise enhanced under these conditions, while the expression strengths of the remaining Moco-biosynthesis genes were not up-regulated. This finding underlines the results of [50], who suggest that in Escherichia coli, the NIT-7A homolog MoaA catalyzes the rate-determining step in cPMP synthesis ( Figure 8A), as documented by the finding that the k cat for the conversion of 3´,8-cH 2 GTP into cPMP is approximately 4-fold higher than the k cat determined for the conversion of GTP to 3´,8-cH 2 GTP [8,9]. Figure 2).…”

DMSO Reductase Family: Phylogenetics and Applications of Extremophiles

“…The coordination and structure of the MoCo in DMSO reductases differentiate them from the rest of molybdoenzymes and tungsten-enzymes families and give to these enzymes their specific functionalities. However, despite the existence of a wide range of cofactors in molybdoenzymes and tungsto-enzymes, the first three reactions for the biosynthesis of the molybdenum cofactor are shared among all families [1,3,16,17]. DMSO reductases experience further modifications in the final steps after pyranopterin formation to achieve the formation of the bis-molybdopterin-guanine dinucleotide (Bis-MGD).…”

“…MoaA is a member of the radical S -adenosyl- l -methionine (SAM) superfamily and possess two [4Fe-4S] clusters (one in each monomer), which are oxygen sensitive [16]. MoaA is the first enzyme to act and it is responsible for forming the unstable and oxygen-sensitive intermediate 3′,8-cyclo-7,8-dihydro-guanosine 5′-triphosphate (3′,8-cH2GTP) (not shown in Figure 2) through a complex rearrangement of the 5′-GTP molecule [3,16,17,18,19]. MoaC acts in the conversion of 3′,8-cH2GTP to cPMP (the pyranopterin backbone).…”

The Requirement of Inorganic Fe-S Clusters for the Biosynthesis of the Organometallic Molybdenum Cofactor

“…Especially in recent years, it has become obvious that most reaction pathways for the synthesis of complex metal-containing cofactors have recruited radical/SAM chemistry [14]. One example has been the identification of the mechanism of the radical/SAM enzyme MoaA, a GTP 3 ,8-cyclase in the biosynthesis of Moco of the diverse class of molybdoenzymes [15,16]. The number of known radical/SAM-dependent enzymes grew exponentially during the last years, with an initial identification of 600 members of the superfamily by Sofia et al in 2001 that until today has increased to more than 113,000 members [14].…”