Identification of enzymes homologous to isocitrate dehydrogenase that are involved in coenzyme B and leucine biosynthesis in methanoarchaea

Two putative Methanococcus jannaschii isocitrate dehydrogenase genes, MJ159 6 and MJ0720, were cloned and overexpressed in Escherichia coli, and their gene products were tested for the ability to catalyze the NAD- and NADP-dep endent oxidative decarboxylation of DL-threo-3-isopropylmalic acid, threo-i socitrate, erythroisocitrate, and homologs of threo-isocitrate. Neither enz yme was found to use any of the isomers of isocitrate as a substrate. The p rotein product of the MJ1596 gene, designated AksF, catalyzed the NAD-depen dent decarboxylation of intermediates in the biosynthesis of 7-mercaptohept anoic acid, a moiety of methanoarchaeal coenzyme B (7-mercaptoheptanylthreo nine phosphate). These intermediates included (-)-threo-isohomocitrate [(-) -threo-1-hydroxy-1,2,4-butanetricarboxylic acid], (-)-threo-iso(homo)(2)cit rate [(-)-threo-1-hydroxy- 1,2,5-pentanetricarboxylic acid], and (-)-threo- iso (homo)(3)citrate [(-) -threo-1-hydroxy-1,2,6-hexanetricarboxylic acid]. The protein product of MJ0720 was found to be alpha-isopropytmalate dehydr ogenase (LeuB) and was found to catalyze the NAB-dependent decarboxylation of one isomer of DL-threo-isopropylmalate to 2-ketoisocaproate; thus, it is involved in the biosynthesis of leucine. The AksF enzyme proved to be ther mostable, losing only 10% of its enzymatic activity after heating at 100 de grees C for 10 min, whereas the LeuB enzyme lost 50% of its enzymatic activ ity after heating at 80 degrees C for 10 min.