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Isoleucine Biosynthesis in Leptospira interrogans Serotype lai Strain 56601 Proceeds via a Threonine-Independent Pathway ^,

Laboratory of Microbial Molecular Physiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032,¹ State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100,² Department of Microbiology and Parasitology, Shanghai Second Medical University, Shanghai 200025,³ Chinese National Human Genome Center at Shanghai, Shanghai 201203, China,⁴ F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland⁵

Received 2 February 2004/ Accepted 6 May 2004

Three leuA-like protein-coding sequences were identified in Leptospira interrogans. One of these, the cimA gene, was shown to encode citramalate synthase (EC 4.1.3.-). The other two encoded -isopropylmalate synthase (EC 4.1.3.12). Expressed in Escherichia coli, the citramalate synthase was purified and characterized. Although its activity was relatively low, it was strictly specific for pyruvate as the keto acid substrate. Unlike the citramalate synthase of the thermophile Methanococcus jannaschii, the L. interrogans enzyme is temperature sensitive but exhibits a much lower K_m (0.04 mM) for pyruvate. The reaction product was characterized as (R)-citramalate, and the proposed ß-methyl-D-malate pathway was further confirmed by demonstrating that citraconate was the substrate for the following reaction. This alternative pathway for isoleucine biosynthesis from pyruvate was analyzed both in vitro by assays of leptospiral isopropylmalate isomerase (EC 4.2.1.33) and ß-isopropylmalate dehydrogenase (EC 1.1.1.85) in E. coli extracts bearing the corresponding clones and in vivo by complementation of E. coli ilvA, leuC/D, and leuB mutants. Thus, the existence of a leucine-like pathway for isoleucine biosynthesis in L. interrogans under physiological conditions was unequivocally proven. Significant variations in either the enzymatic activities or mRNA levels of the cimA and leuA genes were detected in L. interrogans grown on minimal medium supplemented with different levels of the corresponding amino acids or in cells grown on serum-containing rich medium. The similarity of this metabolic pathway in leptospires and archaea is consistent with the evolutionarily primitive status of the eubacterial spirochetes.

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Dedicated to late professor emeritus H. E. Umbarger (Purdue University, Lafayette, Ind.) in recognition of his long-time education, care, and help to G.P.Z. while he was studying at Purdue University during the 1980s and for his lifetime contributions to the study of branched-chain amino acid biosynthesis in bacteria.

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