β-Decarboxylating dehydrogenases comprise 3-isopropylmalate dehydrogenase, isocitrate dehydrogenase, and homoisocitrate dehydrogenase. They share a high degree of amino acid sequence identity and occupy equivalent positions in the amino acid biosynthetic pathways for leucine, glutamate, and lysine, respectively. Therefore, not only the enzymes but also the whole pathways should have evolved from a common ancestral pathway, In Pyrococcus horikoshii, only one pathway of the three has been identified in the genomic sequence, and PH1722 is the sole β-decarboxylating dehydrogenase gene. The organism does not require leucine, glutamate, or lysine for growth; the single pathway might play multiple (i.e., ancestral) roles in amino acid biosynthesis. The PH1722 gene was cloned and expressed in Escherichia coli and the substrate specificity of the recombinant enzyme was investigated. It exhibited activities on isocitrate and homoisocitrate at near equal efficiency, but not on 3-isopropylmalate. PHI 722 is thus a novel, bifunctional β-decarboxylating dehydrogenase, which likely plays a dual role in glutamate and lysine biosynthesis in vivo. © 2005 Elsevier Inc. All rights reserved.