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The recent discovery of a coenzyme B12-dependent acyl-coenzyme A (acyl-CoA) mutase isomerizing 3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in the mesophilic bacterium Aquincola tertiaricarbonis L108 (N. Yaneva, J. Schuster, F. Schäfer, V. Lede, D. Przybylski, T. Paproth, H. Harms, R. H. Müller, and T. Rohwerder, J Biol Chem 287:15502-15511, 2012, http://dx.doi.org/10.1074/jbc.M111.314690) 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.

applied and environmental microbiology

Thermophilic Coenzyme B <sub>12</sub> -Dependent Acyl Coenzyme A (CoA) Mutase from Kyrpidia tusciae DSM 2912 Preferentially Catalyzes Isomerization of ( <i>R</i> )-3-Hydroxybutyryl-CoA and 2-Hydroxyisobutyryl-CoA

Thermophilic Coenzyme B 12 -Dependent Acyl Coenzyme A (CoA) Mutase from Kyrpidia tusciae DSM 2912 Preferentially Catalyzes Isomerization of ( R )-3-Hydroxybutyryl-CoA and 2-Hydroxyisobutyryl-CoA

Thermophilic Coenzyme B ₁₂ -Dependent Acyl Coenzyme A (CoA) Mutase from Kyrpidia tusciae DSM 2912 Preferentially Catalyzes Isomerization of ( R )-3-Hydroxybutyryl-CoA and 2-Hydroxyisobutyryl-CoA