© 2005 Heron Publishing—Victoria, Canada
Characterization of an archaeal malic enzyme from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1
Wakao Fukuda (1), Yulia Sari Ismail (1), Toshiaki Fukui (1, 3), Haruyuki Atomi (1) and Tadayuki Imanaka (1, 3)
1. Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan / 2. Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan / 3. Corresponding author ([email protected]) / Received September 9, 2005; accepted December 16, 2004; published online January 6, 2005
Although the interconversion between C4 and C3 compounds has an important role in overall metabolism, limited information is available on the properties and regulation of enzymes acting on these metabolites in hyperthermophilic archaea. Malic enzyme is one of the enzymes involved in this interconversion, catalyzing the oxidative decarboxylation of malate to pyruvate as well as the reductive carboxylation coupled with NAD(P)H. This study focused on the enzymatic properties and expression profile of an uncharacterized homolog of malic enzyme identified in the genome of a heterotrophic, hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-Mae). The amino acid sequence of Tk-Mae was 52–58% identical to those of malic enzymes from bacteria, whereas the similarities to the eukaryotic homologs were lower. Several catalytically important regions and residues were conserved in the primary structure of Tk-Mae. The recombinant protein, which formed a homodimer, exhibited thermostable malic enzyme activity with strict divalent cation dependency. The enzyme preferred NADP+ rather than NAD+, but did not catalyze the decarboxylation of oxaloacetate, unlike the usual NADP-dependent malic enzymes. The apparent Michaelis constant (Km) of Tk-Mae for malate (16.9 mM) was much larger than those of known enzymes, leading to no strong preference for the reaction direction. Transcription of the gene encoding Tk-Mae and intracellular malic enzyme activity in T. kodakaraensis were constitutively weak, regardless of the growth substrates. Possible roles of Tk-Mae are discussed based on these results and the metabolic pathways of T. kodakaraensis deduced from the genome sequence.
Keywords: carbon metabolism, hyperthermophile, malate, pyruvate, tricarboxylic acid cycle.
ISSN 1472-3654 (Online) ISSN 1472-3646 (Print) Copyright © 2002–2007 Heron Publishing