Biochemical characterization of a new 7,8‐dihydro‐6‐hydroxymethylpterin pyrophosphokinase (HPPK) from Methanocaldococcus jannaschii

Abstract

Methanogenic archaea are strict anaerobes that produce methane as a byproduct of their essential energy metabolism known as methanogenesis. Tetrahydromethanopterin (H4MPT), a specialized cofactor similar to tetrahydrofolate (H4F), is a C1carrier required for the methanogenesis pathway. The biosynthesis of H4MPT utilizes a similar series of reactions used in H4F biosynthesis, but they are catalyzed by nonorthologous enzymes. Here, we report the recombinant expression, purification, and enzymatic properties of MptE (MJ1634), a novel 7,8‐dihydro‐6‐hydroxymethylpterin pyrophosphokinase (HPPK) identified to catalyze the final step in the biosynthesis of the pterin portion in H4MPT in Methancaldococcus jannaschii by catalyzing the transfer of a pyrophosphate from ATP to the substrate dihydro‐6‐hydroxymethylpterin (H2HMP). This novel HPPK shows a distinctive sequence from the bacterial counterpart; instead, it belongs to the superfamily of thiamine pyrophosphokinase (TPK) catalytic domain. The gene encoding MptE from M. jannaschii was overexpressed in Escherichia coli, and the his‐tagged recombinant enzyme was purified by metal‐affinity chromatography. After purification, initial enzyme assays were performed and analyzed via HPLC, which revealed a similar catalytic efficiency to bacterial HPPKs. Site‐directed mutagenesis studies coupled with structural analyses revealed that Asp106 and Asp171 are essential for Mg2+ coordination, His129 and Phe169 play important roles in H2HMP binding, and Lys217 is important for transition state stabilization. These residues were selected because they are strictly conserved in the archaeal protein family COG1634 or thiamine pyrophosphokinase (TPK), whose active site resembles those in the COG1634 family. Currently, kinetic studies are underway for additional variants to further identify key residues of structural and catalytic importance.