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J. Biol. Chem., Vol. 278, Issue 3, 1864-1871, January 17, 2003

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Characterization of Homoisocitrate Dehydrogenase Involved in Lysine Biosynthesis of an Extremely Thermophilic Bacterium, Thermus thermophilus HB27, and Evolutionary Implication of -Decarboxylating Dehydrogenase^*

Junichi Miyazaki, Nobuyuki Kobashi, Makoto Nishiyama^§, and Hisakazu Yamane

From the Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Although the presence of an enzyme that catalyzes -decarboxylating dehydrogenation of homoisocitrate to synthesize 2-oxoadipate has been postulated in the lysine biosynthesis pathway through -aminoadipate (AAA), the enzyme has not yet been analyzed at all, because no gene encoding the enzyme has been identified until recently. A gene encoding a protein with a significant amino acid sequence identity to both isocitrate dehydrogenase and 3-isopropylmalate dehydrogenase was cloned from Thermus thermophilus HB27. The gene product produced in recombinant Escherichia coli cells demonstrated homoisocitrate dehydrogenase (HICDH) activity. A knockout mutant of the gene showed an AAA-auxotrophic phenotype, indicating that the gene product is involved in lysine biosynthesis through AAA. We therefore named this gene hicdh. HICDH, the gene product, did not catalyze the conversion of 3-isopropylmalate to 2-oxoisocaproate, a leucine biosynthetic reaction, but it did recognize isocitrate, a related compound in the tricarboxylic acid cycle, as well as homoisocitrate as a substrate. It is of interest that HICDH catalyzes the reaction with isocitrate about 20 times more efficiently than the reaction with the putative native substrate, homoisocitrate. The broad specificity and possible dual function suggest that this enzyme represents a key link in the evolution of the pathways utilizing citrate derivatives. Site-directed mutagenesis study reveals that replacement of Arg⁸⁵ with Val in HICDH causes complete loss of activity with isocitrate but significant activity with 3-isopropylmalate and retains activity with homoisocitrate. These results indicate that Arg⁸⁵ is a key residue for both substrate specificity and evolution of -decarboxylating dehydrogenases.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB075751, AB085838, and AB085839.

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