Biosynthesis of 6-Thioguanine: Characterizing Two Intermediates Involved in Its Thioamide Formation Reaction

6-Thioguanosine 5′-monophosphate (6-TGMP) is the biologically active nucleotide form of the antimetabolite 6-thioguanine produced by Erwinia amylovorans. The YcfA-YcfC enzymatic pair catalyzes the incorporation of the thioamide functional group in GMP via an oxygen-to-sulfur replacement process. YcfA is a member of the adenine nucleotide alpha hydrolase-like (AANH-like) superfamily, and YcfC is a pyridoxal 5′-phosphate (PLP)-dependent enzyme. Two mechanistic models exist, depending on whether YcfC is a C–S lyase or a cysteine desulfurase, to explain the trans-sulfuration reaction. Herein, we investigate the YcfA-YcfC trans-sulfuration reaction using anaerobically purified YcfA and YcfC enzymes in the absence of reductants. Steady state and presteady state kinetic studies suggest the mechanism involves two distinct intermediates and differs from the common trans-sulfuration pathway used in, for example, tRNA thionucleotide biosynthesis.