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 coil, and their recombinant proteins, Tk-G3PDH, Tk-NOX and Tk-MOX, were characterized. The optimal temperature of Tk-G3PDH for activity was 80 degrees 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 x 10(3) M(-1)s(-1) at 80 degrees C, compared with 9.83 x 10(5) M(-1)s(-1) for the E. coli G3PDH complex at 37 degrees C. Interestingly, TicG3PDH can catalyze this reaction even as a monomer, whereas GIpA must form a complex with lIpB 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. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.