Anaerobic glycerol-3-phosphate dehydrogenase complex from hyperthermophilic archaeon Thermococcus kodakarensis KOD1

Glycerol-3-phosphate (G3P) is a key intermediate of glycerol metabolism and is oxidized to dihydroxyacetone phosphate aerobically or anaerobically by appropriate G3P dehydrogenases. A hyperthermophilic archaeon Thermococcus kodakarensis KOD1 has a novel operon consisting of three genes encoding an anaerobic G3P dehydrogenase (G3PDH), an NADH oxidase (NOX), and a molybdopterin oxidoreductase (MOX). Typically, the G3PDH gene (glpA) is included in an operon with genes encoding essential subunits of the G3PDH complex, glpB and glpC. The three genes from T. kodakarensis were cloned and expressed in Escherichia coli, and their recombinant proteins, Tk-G3PDH, Tk-NOX and Tk-MOX, were characterized. The optimal temperature of Tk-G3PDH for activity was 80°C, indicating high thermal stability. Tk-G3PDH has flavin adenine dinucleotide as a prosthetic group and catalyzes oxidation of G3P with k_cat/K_m 1.93 × 10³ M^-1s^-1 at 80°C, compared with 9.83 × 10⁵ M^-1s^-1 for the E. coli G3PDH complex at 37°C. Interestingly, Tk-G3PDH can catalyze this reaction even as a monomer, whereas GlpA must form a complex with GlpB and GlpC. Tk-G3PDH also forms a putative heteropentamer with Tk-NOX and Tk-MOX (G3PDH:NOX:MOX = 2:2:1). This complex may form an electron transfer pathway to a final electron acceptor in the cell membrane, as is the case for the typical G3PDH complex GlpABC.