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

Abstract

) 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...

Production of 2-Hydroxyisobutyric Acid from Methanol by Methylobacterium extorquens AM1 Expressing ( R )-3-Hydroxybutyryl Coenzyme A-Isomerizing Enzymes

“…Consequently, both enzymes equally favor the (R)-enantiomer above the (S)-enantiomer of 3-hydroxybutyryl-CoA. Nevertheless, we clearly characterized RCM Bmas as a mesophilic enzyme showing a temperature optimum of 35°C, while RCM Ktus has its activity maximum at 55°C (12). This thermal adaptation may be caused by various molecular mechanisms, such as the number of salt bridges and hydrophobic interactions (27).…”

“…Therefore, the RCM from B. massiliosenegalensis JC6 (RCM Bmas ) was considered to be mesophilic. Subsequently, the values of the kinetic parameters for all three acyl-CoA substrates were determined at 35°C (Table 1) and compared to the values of the kinetic parameters for RCM Ktus previously obtained at 55°C (12). Likely due to the different thermal adaptation, with the activity being about 220 nmol min Ϫ1 mg Ϫ1 for both (R)-3-hydroxybutyryl-and Fig.…”

“…In this connection, a potentially mesophilic RCM candidate was found in the bacterium Bacillus massiliosenegalensis JC6, which was reported to grow well at 37°C (13). This enzyme and the RCM from K. tusciae DSM 2912 share 78.2%, 77.4%, and 52.8% sequence identity with the large substrate-binding mutase subunit RcmA, the small coenzyme B 12 -binding subunit RcmB, and the associated putative G protein chaperone MeaH, respectively (12). Furthermore, the RCM genes are arranged in an identical operonic structure.…”

“…HCM was originally found in Aquincola tertiaricarbonis L108, a bacterial strain capable of degrading the fuel oxygenate methyl tert-butyl ether (5). It was classified as a coenzyme B 12 -dependent acyl-CoA mutase and shown to specifically isomerize 3-hydroxybutyryl-CoA into 2-hydroxyisobutyryl-CoA (3). On the basis of this reaction, the polyhydroxybutyrate (PHB) pathway, a prevalent overflow metabolic pathway of bacteria (6), could be employed for biotechnological 2-HIBA production (7).…”

“…However, product yields were very low in these early studies, an observation that was subsequently ascribed to the fact that HCM from Aquincola tertiaricarbonis L108 is highly specific for (S)-3-hydroxybutyryl-CoA and thus poorly compatible with the PHB cycle (3). In the search for an (R)-3-hydroxybutyryl-CoA-specific coenzyme B 12 -dependent mutase (RCM), a candidate isolated from the thermophilic knallgas bacterium Kyrpidia tusciae DSM 2912 was characterized and could indeed be shown to favor the (R)-isomer (12). After the concomitant heterologous expression of the PhaA, PhaB, and RCM genes, the formation of 2-HIBA could be successfully demonstrated in Escherichia coli strains, but the final titers did not exceed 70 mg liter Ϫ1 .…”