Background: Bacterial B 12 -dependent 2-hydroxyisobutyryl-CoA mutase specifically catalyzes the isomerization of (S)-3-hydroxybutyryl-and 2-hydroxyisobutyryl-CoA. Results: The crystal structure of 2-hydroxyisobutyryl-CoA mutase shows decisive differences in the active site when compared with the well studied methylmalonyl-CoA mutase. Conclusion: Specificity toward (S)-3-hydroxybutyryl-CoA strongly depends on the active site amino acid Asp A117 . Significance: This is the first structural characterization of a B 12 -dependent mutase with ␣ 2  2 organization isomerizing 2-hydroxyisobutyryl-CoA.
) could pave the way for a complete biosynthesis route to the building block chemical 2-hydroxyisobutyric acid from renewable carbon. However, the enzyme catalyzes only the conversion of the stereoisomer (S)-3-hydroxybutyryl-CoA at reasonable rates, which seriously hampers an efficient combination of mutase and well-established bacterial poly-(R)-3-hydroxybutyrate (PHB) overflow metabolism. Here, we characterize a new 2-hydroxyisobutyryl-CoA mutase found in the thermophilic knallgas bacterium Kyrpidia tusciae DSM 2912. Reconstituted mutase subunits revealed highest activity at 55°C. Surprisingly, already at 30°C, isomerization of (R)-3-hydroxybutyryl-CoA was about 7,000 times more efficient than with the mutase from strain L108. The most striking structural difference between the two mutases, likely determining stereospecificity, is a replacement of active-site residue Asp found in strain L108 at position 117 with Val in the enzyme from strain DSM 2912, resulting in a reversed polarity at this binding site. Overall sequence comparison indicates that both enzymes descended from different prokaryotic thermophilic methylmalonyl-CoA mutases. Concomitant expression of PHB enzymes delivering (R)-3-hydroxybutyryl-CoA (beta-ketothiolase PhaA and acetoacetyl-CoA reductase PhaB from Cupriavidus necator) with the new mutase in Escherichia coli JM109 and BL21 strains incubated on gluconic acid at 37°C led to the production of 2-hydroxyisobutyric acid at maximal titers of 0.7 mM. Measures to improve production in E. coli, such as coexpression of the chaperone MeaH and repression of thioesterase II, are discussed.
Carbon skeleton rearrangement of carboxylic acids via a chemically challenging radical mechanism is catalyzed by coenzyme B 12 -dependent acyl-coenzyme A (acyl-CoA) mutases (1). During catalysis, both acyl-CoA and B 12 molecules are completely buried within the enzyme. This extensive interaction is mediated by highly conserved amino acid residues, forming a characteristic triose phosphate isomerase (TIM) barrel and a Rossman fold. The best-studied member of this enzyme family is methylmalonylCoA mutase (MCM), specifically catalyzing the isomerization of succinyl-and (R)-methylmalonyl-CoA (2). Several genetic defects impairing mitochondrial MCM activity are associated with methylmalonic aciduria, an inborn error of branched-chain amino acid metabolism (3, 4). Another mutase playing a role in central carbon metabolism is ethylmalonyl-CoA mutase (ECM), involved in acetic acid assimilation in bacteria lacking the glyoxylate cycle (5). In addition, isobutyryl-CoA mutase (ICM) appears to function mainly in secondary metabolism, e.g., the bacterial synthesis of polyketide antibiotics (6). Recently, a fourth subfamily of coenzyme B 12 -dependent acyl-CoA mutases has been characterized, specifically catalyzing the interconversion of 3-hydroxybutyrylCoA enantiomers and 2-hydroxyisobutyryl-CoA (7) (Fig. 1). Initially, the 2-hydroxyisobutyryl-CoA mutase (HCM) has been discovered in the bacterial strains Aquincola tertiaricarbon...
Aerobic anoxygenic photosynthesis (AAP) is found in an increasing number of proteobacterial strains thriving in ecosystems ranging from extremely oligotrophic to eutrophic. Here, we have investigated whether the fuel oxygenate-degrading betaproteobacterium Aquincola tertiaricarbonis L108 can use AAP to compensate kinetic limitations at low heterotrophic substrate fluxes. In a fermenter experiment with complete biomass retention and also during chemostat cultivation, strain L108 was challenged with extremely low substrate feeding rates of tert-butyl alcohol (TBA), an intermediate of methyl tert-butyl ether (MTBE). Interestingly, formation of photosynthetic pigments, identified as bacteriochlorophyll a and spirilloxanthin, was only induced in growing cells at TBA feeding rates less than or equal to maintenance requirements observed under energy excess conditions. Growth continued at rates between 0.001 and 0.002 h "1 even when the TBA feed was decreased to values close to 30 % of this maintenance rate. Partial sequencing of genomic DNA of strain L108 revealed a bacteriochlorophyll synthesis gene cluster (bchFNBHL) and photosynthesis regulator genes (ppsR and ppaA) typically found in AAP and other photosynthetic proteobacteria. The usage of light as auxiliary energy source enabling evolution of efficient degradation pathways for kinetically limited heterotrophic substrates and for lowering the threshold substrate concentration S min at which growth becomes zero is discussed.
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