Biosynthesis of isoleucine in methanogenic bacteria: a carbon-13 NMR study

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 ␤-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 ␤-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.

mentioning

confidence: 99%

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

Zhang

Guo

et al. 2004

“…While 13C-labeling studies of whole cells of methanogenic archaebacteria have provided evidence for the common pathway, only the properties of the methanococcal AHAS have been described (11,12,42). The methanococci are marine methanogens which use H2-CO2 or formate as a substrate for methanogenesis (17).…”

mentioning

confidence: 99%

Characterization of enzymes of the branched-chain amino acid biosynthetic pathway in Methanococcus spp

Xing

Whitman

1991

Methanococcus aeolicus, Methanococcus maripaludis, and Methanococcus voltae contain similar levels of four enzymes of branched-chain amino acid biosynthesis: acetohydroxy acid synthase, acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and transaminase B. Following growth at low partial pressures of H2-CO2, the levels of these enzymes in extracts of M. voltae are reduced three-to fivefold, which suggests that their synthesis is regulated. The enzymes from M. aeolicus were found to be similar to the eubacterial and eucaryotic enzymes with respect to molecular weights, pH optima, kinetic properties, and sensitivities to 02. The acetohydroxy acid isomeroreductase has a specific requirement for Mg2+, and other divalent cations were inhibitory. It was stimulated threefold by K+ and NH4+ ions and was able to utilize NADH as well as NADPH.The partially purified enzyme was not sensitive to 2. The dihydroxy acid dehydratase is extremely sensitive to 02and it has a half-iffe under 5% 02 of 6 min at 25°C. Divalent cations were required for activity, and Mg2+, Mn2+, Ni2+, Co2+, and Fe2+ were nearly equally effective. In conclusion, the archaebacterial enzymes are functionally homologous to the eubacterial and eucaryotic enzymes, which implies that this pathway is very ancient.The biosynthesis of the branched-chain amino acids (BCAAs), valine, isoleucine, and leucine, occurs by a parallel set of reactions that is similar in the eubacteria and eucaryotes (for reviews see references 37 and 38 Preparation of extracts and partial purification of enzymes. Extracts of the methanococci were prepared by thawing frozen cells in anaerobic lysis buffer containing 25 mM PIPES [potassium piperazine-N,N'-bis(2-ethanesulfonic acid), pH 6.8], 1 mM dithiothreitol, 1 mM cysteine hydrochloride, and pancreatic DNase (1 mg/100 ml of cell suspension) and centrifuging at 39,000 x g for 20 min at 4°C (42). Freezethawing of these cells was sufficient to disrupt the protein cell wall and produce cell-free extracts. For some experiments, unfrozen extracts were prepared by suspending cells in the anaerobic lysis buffer immediately after harvesting. In buffers containing low concentrations of Na+, these cells rapidly lysed. However, the yield of extract was slightly lower than in extracts prepared by freezing. Dialyzed extracts were prepared as described previously (42). Oxygen was removed from the buffers by flushing with N2 gas for at least 60 min, and strictly anaerobic procedures were used for all purification steps. Chromatography procedures were performed in an anaerobic chamber (Coy Laboratory Products, Ann Arbor, Mich.) in a 5°C cold room. Plastic centrifuge tubes were incubated for at least 24 h in the anaerobic chamber before use to remove dissolved oxygen.After centrifugation, saturated ammonium sulfate (pH 7.5) in water was added slowly to 45 ml of extract at 5°C. The final concentration of ammonium sulfate was 50% of saturation. The solution was mixed slowly for 20 min and centrifuged for 30 min at 30,000 x g at 4°C. The super...

“…The labeling pattern of squalene supported the above conclusions. Based on these data, a general scheme is proposed to account for the contribution of lysine-to-lipid biosynthesis.Our previous papers on the application of the 13C-nuclear magnetic resonance (NMR) technique to study the metabolic pathways of methanogenic bacteria (7,8,10) showed several significant differences in biosynthesis when compared with the biosynthesis of other amino acids by bacteria and eucaryotes. Similar studies for comparison with halobacteria are lacking.…”

mentioning

confidence: 99%

Mevalonic acid is partially synthesized from amino acids in Halobacterium cutirubrum: a 13C nuclear magnetic resonance study

Ekiel¹,

Sprott²,

Smith³

1986

13C nuclear magnetic resonance revealed an unusual pathway for the biosynthesis of lipids in Halobacterium cutirubrum and H. halobium. Mevalonic acid was not synthesized from three acetyl-coenzyme A molecules, as has been suggested previously, and the branch-methyl and methine carbons in phytanyl chains were derived from neither acetate nor glycerol. Instead, they were supplied by the degradation of amino acids, in particular of lysine. Presumably, two different types of two-carbon fragments were used simultaneously by halobacteria for the biosynthesis of mevalonate. The labeling pattern of squalene supported the above conclusions. Based on these data, a general scheme is proposed to account for the contribution of lysine-to-lipid biosynthesis.Our previous papers on the application of the 13C-nuclear magnetic resonance (NMR) technique to study the metabolic pathways of methanogenic bacteria (7,8,10) showed several significant differences in biosynthesis when compared with the biosynthesis of other amino acids by bacteria and eucaryotes. Similar studies for comparison with halobacteria are lacking. Halobacteria are archaebacteria also, but with an aerobic rather than anaerobic mode of metabolism.Halobacteria require quite complicated media for growth, and, in contrast to the aceticlastic methanogens, acetate can be used for growth only when combined with several additional carbon sources.