Isoleucine biosynthesis in Leptospira interrogans serotype lai strain 56601 proceeds via a threonine-independent pathway.
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 alpha-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 beta-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 beta-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.
Laboratory of Microbial Molecular Physiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
Hai
H
Xu
Yuzhen
Y
Zhang
Xiaokui
X
Guo
Shuangxi
S
Ren
Andreas A
AA
Staempfli
Juishen
J
Chiao
Weihong
W
Jiang
Guoping
G
Zhao
D015079:2-Isopropylmalate Synthase / Q000235:genetics / Q000378:metabolism; D050539:3-Isopropylmalate Dehydrogenase; D000222:Adaptation, Physiological / Q000235:genetics / Q000502:physiology; D000429:Alcohol Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology; D005798:Genes, Bacterial / Q000235:genetics / Q000502:physiology; D005816:Genetic Complementation Test; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism; D007532:Isoleucine / Q000096:biosynthesis*; D007535:Isomerases / Q000302:isolation & purification / Q000378:metabolism; D007921:Leptospira interrogans / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000096:biosynthesis; D017017:Methanococcus / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D019289:Pyruvic Acid / Q000378:metabolism; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism*
D001426:Bacterial Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007532:Isoleucine; D013912:Threonine; D019289:Pyruvic Acid; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase; D015079:2-Isopropylmalate Synthase; D006836:Hydro-Lyases; C032241:isopropylmalate isomerase; C020052:dihydroxyacid dehydratase; D007535:Isomerases; D007930:Leucine