Mesaconyl-coenzyme A hydratase, a new enzyme of two central carbon metabolic pathways in bacteria

Abstract

The coenzyme A (CoA)-activated C5-dicarboxylic acids mesaconyl-CoA and beta-methylmalyl-CoA play roles in two as yet not completely resolved central carbon metabolic pathways in bacteria. First, these compounds are intermediates in the 3-hydroxypropionate cycle for autotrophic CO2 fixation in Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium. Second, mesaconyl-CoA and beta-methylmalyl-CoA are intermediates in the ethylmalonyl-CoA pathway for acetate assimilation in various bacteria, e.g., in Rhodobacter sphaeroides, Methylobacterium extorquens, and Streptomyces species. In both cases, mesaconyl-CoA hydratase was postulated to catalyze the interconversion of mesaconyl-CoA and beta-methylmalyl-CoA. The putative genes coding for this enzyme in C. aurantiacus and R. sphaeroides were cloned and heterologously expressed in Escherichia coli, and the proteins were purified and studied. The recombinant homodimeric 80-kDa proteins catalyzed the reversible dehydration of erythro-beta-methylmalyl-CoA to mesaconyl-CoA with rates of 1,300 micromol min(-1) mg protein(-1). Genes coding for similar enzymes with two (R)-enoyl-CoA hydratase domains are present in the genomes of Roseiflexus, Methylobacterium, Hyphomonas, Rhodospirillum, Xanthobacter, Caulobacter, Magnetospirillum, Jannaschia, Sagittula, Parvibaculum, Stappia, Oceanicola, Loktanella, Silicibacter, Roseobacter, Roseovarius, Dinoroseobacter, Sulfitobacter, Paracoccus, and Ralstonia species. A similar yet distinct class of enzymes containing only one hydratase domain was found in various other bacteria, such as Streptomyces species. The role of this widely distributed new enzyme is discussed.

FIG. 1.

Proposed roles of mesaconyl-CoA hydratase/β-methylmalyl-CoA dehydratase in two bacterial pathways of central carbon metabolism. The boxes mark the postulated reactions catalyzed by the enzyme. (A) Proposed 3-hydroxypropionate cycle of autotrophic CO₂ fixation in C. aurantiacus. 1, acetyl-CoA carboxylase; 2, malonyl-CoA reductase; 3, propionyl-CoA synthase; 4, propionyl-CoA carboxylase; 5, methylmalonyl-CoA epimerase; 6, methylmalonyl-CoA mutase; 7, succinyl-CoA:l-malate CoA transferase; 8, succinate dehydrogenase; 9, fumarate hydratase; 10, l-malyl-CoA lyase/β-methylmalyl-CoA lyase; 11, mesaconyl-CoA hydratase; 12, unknown reactions; 13, succinyl-CoA:citramalate CoA-transferase; 14, citramalyl-CoA-lyase. (B) Proposed alternate glyoxylate cycle for acetate assimilation in isocitrate-lyase-negative bacteria, such as R. sphaeroides. This pathway, termed the ethylmalonyl-CoA pathway, also serves to assimilate acetyl-CoA produced from methanol and CO₂ in methylotrophic bacteria, such as M. extorquens, which uses the serine cycle for formaldehyde fixation during growth on methanol. 1, β-ketothiolase; 2, acetoacetyl-CoA reductase; 3, crotonase; 4, crotonyl-CoA carboxylase/reductase; 5, ethylmalonyl-CoA mutase; 6, methylsuccinyl-CoA dehydrogenase; 7, mesaconyl-CoA hydratase; 8, l-malyl-CoA lyase/β-methylmalyl-CoA lyase; 9, malyl-CoA hydrolase; 10, propionyl-CoA carboxylase; 11, methylmalonyl-CoA epimerase; 12, methylmalonyl-CoA mutase.

FIG. 2.

Denaturing PAGE (12.5%) of various purification steps of heterologously expressed mesaconyl-CoA hydratase of C. aurantiacus and R. sphaeroides from extracts of 3 g E. coli cells. Lanes 1 and 7, molecular mass standards; lanes 2 to 4, Chloroflexus enzyme; lane 2, extract (100,000 × g supernatant) of induced E. coli cells (45 μg protein); lane 3, heat precipitation fraction (20 μg protein); lane 4, affinity chromatography fraction (8 μg protein); lanes 5 and 6, Rhodobacter enzyme; lane 5, extract (100,000 × g supernatant) of induced E. coli cells (30 μg protein); lane 6, affinity chromatography fraction (8 μg protein). The gel was stained with Coomassie brilliant blue R-250.

FIG. 3.

UV spectra of coenzyme A (^.._..), propionyl-CoA (—), β-methylmalyl-CoA (—), and the product of the hydratase reaction, mesaconyl-CoA (^…….). The spectra were recorded in 40 mM K₂HPO₄/formic acid buffer, pH 4.2, and were normalized to the same absorption at 260 nm.

FIG. 4.

HPLC separation of [¹⁴C]mesaconyl-CoA as the product of the mesaconyl-CoA hydratase from [¹⁴C]β-methylmalyl-CoA and [¹⁴C]propionyl-CoA. [¹⁴C]β-methylmalyl-CoA is enzymatically formed from [¹⁴C]propionyl-CoA and glyoxylate with recombinant l-malyl-CoA/β-methylmalyl-CoA lyase. CoA and its derivatives were detected at 260 nm. Free acids of the corresponding CoA thioesters were eluted between 2 and 5 min. (A) Before addition of glyoxylate to the assay mixture. (B) Ten minutes after addition of glyoxylate. (C) Formation of mesaconyl-CoA after another 10 minutes of incubation with recombinant mesaconyl-CoA hydratase. The sensitivities of ¹⁴C detection by solid-phase scintillation counting in the different runs were the same, and therefore, the signals can be directly compared. For the conditions, see Materials and Methods.

FIG. 5.

Phylogenetic tree of homologues of mesaconyl-CoA hydratase based on amino acid sequences. Note that members of this group of enzymes have approximately double the sizes of normal enoyl-CoA hydratases and contain two enoyl-CoA hydratase domains. They are formed by duplication of genes containing only one enoyl-CoA hydratase domain (tree not shown). The tree topography and evolutionary distances are given by the neighbor-joining method with Poisson correction. The scale bar represents a difference of 0.1 substitution per site. The numbers at the nodes indicate the percentage bootstrap values for the clade of this group in 1,000 replications. Only numbers above 50% were considered to be significant.