Production of mesaconate in Escherichia coli by engineered glutamate mutase pathway.

Mesaconate is an intermediate in the glutamate degradation pathway of microorganisms such as Clostridium tetanomorphum. However, metabolic engineering to produce mesaconate has not been reported previously. In this work, two enzymes involved in mesaconate production, glutamate mutase and 3-methylaspartate ammonia lyase from C. tetanomorphum, were recombinantly expressed in Escherichia coli. To improve mesaconate production, reactivatase of glutamate mutase was discovered and adenosylcobalamin availability was increased. In addition, glutamate mutase was engineered to improve the in vivo activity. These efforts led to efficient mesaconate production at a titer of 7.81 g/L in shake flask with glutamate feeding. Then a full biosynthetic pathway was constructed to produce mesaconate at a titer of 6.96 g/L directly from glucose. In summary, we have engineered an efficient system in E. coli for the biosynthesis of mesaconate.

Molecular modeling studies of the conserved B(12)-binding motif and its variants from Clostridium tetanomorphum glutamate mutase.

The coupling of an aspartate residue with an active site histidine plays a pivotal role in enzyme catalysis. The His-Asp pair in glutamate mutase and other B(12)-dependent mutases is not only responsible for coenzyme-binding, but is also involved in fine-tuning the enzymatic activities. Our modeling results show that the His-Asp pair is arranged in a highly organized manner. Except for carboxymethylated Cys or Glu, a less hindered or non-charged amino acid residue is preferred between the conserved histidine and aspartate residue.

Alteration of the diastereoselectivity of 3-methylaspartate ammonia lyase by using structure-based mutagenesis.

3-Methylaspartate ammonia-lyase (MAL) catalyzes the reversible amination of mesaconate to give both (2S,3S)-3-methylaspartic acid and (2S,3R)-3-methylaspartic acid as products. The deamination mechanism of MAL is likely to involve general base catalysis, in which a catalytic base abstracts the C3 proton of the respective stereoisomer to generate an enolate anion intermediate that is stabilized by coordination to the essential active-site Mg(II) ion. The crystal structure of MAL in complex with (2S,3S)-3-methylaspartic acid suggests that Lys331 is the only candidate in the vicinity that can function as a general base catalyst. The structure of the complex further suggests that two other residues, His194 and Gln329, are responsible for binding the C4 carboxylate group of (2S,3S)-3-methylaspartic acid, and hence are likely candidates to assist the Mg(II) ion in stabilizing the enolate anion intermediate. In this study, the importance of Lys331, His194, and Gln329 for the activity and stereoselectivity of MAL was investigated by site-directed mutagenesis. His194 and Gln329 were replaced with either an alanine or arginine, whereas Lys331 was mutated to a glycine, alanine, glutamine, arginine, or histidine. The properties of the mutant proteins were investigated by circular dichroism (CD) spectroscopy, kinetic analysis, and (1)H NMR spectroscopy. The CD spectra of all mutants were comparable to that of wild-type MAL, and this indicates that these mutations did not result in any major conformational changes. Kinetic studies demonstrated that the mutations have a profound effect on the values of k(cat) and k(cat)/K(M); this implicates Lys331, His194 and Gln329 as mechanistically important. The (1)H NMR spectra of the amination and deamination reactions catalyzed by the mutant enzymes K331A, H194A, and Q329A showed that these mutants have strongly enhanced diastereoselectivities. In the amination direction, they catalyze the conversion of mesaconate to yield only (2S,3S)-3-methylaspartic acid, with no detectable formation of (2S,3R)-3-methylaspartic acid. The results are discussed in terms of a mechanism in which Lys331, His194, and Gln329 are involved in positioning the substrate and in formation and stabilization of the enolate anion intermediate.

Interactions between coenzyme B12 analogs and adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum.

Adenosylcobalamin (AdoCbl)-dependent glutamate mutase from Clostridium tetanomorphum comprises two weakly-associating subunits, MutS and MutE, which combine with AdoCbl to form the active holo-enzyme. Three coenzyme analogs, methylcobinamide (MeCbi), adenosylcobinamide (AdoCbi) and adeosylcobinamide-GDP (AdoCbi-GDP), were synthesized at milligram scale. Equilibrium dialysis was used to measure the binding of coenzyme B(12) analogs to glutamate mutase. Our results show that, unlike AdoCbl-dependent methylmalonyl CoA mutase, the ratio k(cat)/K(m) decreased approximately 10(4)-fold in both cases when AdoCbi or AdoCbi-GDP was used as the cofactor. The coenzyme analog-binding studies show that, in the absence of the ribonucleotide tail of AdoCbl, the enzyme's active site cannot correctly accommodate the coenzyme analog AdoCbi. The results presented here shed some light on the cobalt-carbon cleavage mechanism of B(12).