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Published on: February 15, 2016
Biosynthesis of 6-thioguanine: characterizing two intermediates involved in its thioamide formation reaction
Kelin Li1, Juan Lopez1, Zion Kang1
1Department of Chemistry, Boston University, Boston, MA 02215, USA.
Abstract:
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 YfcA and YcfC enzymes in the absence of reductants. Steady state and pre-steady 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.
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