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Journal of Bacteriology, September 2000, p. 5013-5016, Vol. 182, No. 17
Department of Biochemistry (0308), Virginia
Polytechnic Institute and State University, Blacksburg, Virginia
24061
Received 4 February 2000/Accepted 6 June 2000
Two putative Methanococcus jannaschii isocitrate
dehydrogenase genes, MJ1596 and MJ0720, were cloned and overexpressed
in Escherichia coli, and their gene products were tested
for the ability to catalyze the NAD- and NADP-dependent oxidative
decarboxylation of DL-threo-3-isopropylmalic
acid, threo-isocitrate, erythro-isocitrate, and
homologs of threo-isocitrate. Neither enzyme was found to use any of the isomers of isocitrate as a substrate. The protein product of the MJ1596 gene, designated AksF, catalyzed the
NAD-dependent decarboxylation of intermediates in the biosynthesis of
7-mercaptoheptanoic acid, a moiety of methanoarchaeal coenzyme B
(7-mercaptoheptanylthreonine phosphate). These intermediates included
(
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification of Enzymes Homologous to Isocitrate Dehydrogenase
That Are Involved in Coenzyme B and Leucine Biosynthesis in
Methanoarchaea
)-threo-isohomocitrate [()-threo-1-hydroxy-1,2,4-butanetricarboxylic acid],
()-threo-iso(homo)2citrate [()-threo-1-hydroxy-1,2,5-pentanetricarboxylic acid],
and ()-threo-iso(homo)3citrate [()-threo-1-hydroxy-1,2,6-hexanetricarboxylic acid].
The protein product of MJ0720 was found to be 
-isopropylmalate
dehydrogenase (LeuB) and was found to catalyze the NAD-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 thermostable, losing only 10% of its enzymatic activity
after heating at 100°C for 10 min, whereas the LeuB enzyme lost 50%
of its enzymatic activity after heating at 80°C for 10 min.
*
Corresponding author. Mailing address: Department of
Biochemistry (0308), Virginia Polytechnic Institute and State
University, Blacksburg, VA 24061. Phone: (540) 231-6605. Fax: (540)
231-9070. E-mail: rhwhite{at}vt.edu.
Journal of Bacteriology, September 2000, p. 5013-5016, Vol. 182, No. 17
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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