Isoleucine biosynthesis in Leptospira interrogans serotype lai strain 56601 proceeds via a threonine-independent pathway

Abstract

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.

FIG. 1.

Schematic illustration of the leucine-like pyruvate pathway of isoleucine biosynthesis in L. interrogans and the corresponding pathway of leucine biosynthesis.

FIG. 2.

SDS-PAGE analysis of purified CimA (LA2350) protein. CimA was purified as described in Materials and Methods. The amount of purified protein loaded was 4 μg. The molecular size markers were purchased from Promega Corp.

FIG. 3.

Optimal reaction temperature (▪) and thermostability (▴) of L. interrogans citramalate synthase. The experiments were carried out as described in Materials and Methods, with cell extract containing either the highly expressed (A) or the purified (B) enzyme.

FIG. 4.

Chirality of the citramalate produced by LA2350 (CimA) reaction. (a) Peaks of (R)-citramalate and (S)-citramalate as products of the condensation reaction catalyzed by the crude enzyme. Standards were added to the samples (top), highlighting the elution positions of the corresponding R and S isomers. Most of the products were (R)-citramalate. (b) Purified enzyme with TES buffer (imidazole was removed from the sample buffer) was used for the reaction. (R)-citramalate is the predominant product. The peak area of (R)-citramalate was 183,099.1 μV  ·  s (98.57%), while the peak area of (S)-citramalate was 2,647.0 μV  ·  s (1.43%). (c) Purified enzyme with imidazole was used for the reaction. The production of (R)-citramalate was decreased. The peak area of (R)-citramalate was 40,327.0 μV  ·  s (83%), while the peak area of (S)-citramalate was 7,933.6 μV  ·  s (16.44%). These two reactions were carried out under the same conditions, including the same concentrations of enzymes and substrates, except for the presence or absence of imidazole in the reaction buffer.

FIG. 5.

Expression levels of cimA (LA2350, diamond), leuA1 (LA2202, triangle), and leuA2 (LA0469, square) in L. interrogans revealed by RT-PCR. L. interrogans was grown in different media as described in Materials and Methods. Solid lines connecting filled data points represent the conditions under which isoleucine was added to EMJH medium. Dashed lines connecting filled data points represent the leucine-supplemented EMJH medium. The single open unconnected data points represent Korthof medium. All data points represent average measurements from three independently conducted assays with error bars indicated. ct, cycle threshold.