Engineered and Native Coenzyme B12-dependent Isovaleryl-CoA/Pivalyl-CoA Mutase

Abstract

Adenosylcobalamin-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently demonstrated that an isobutyryl-CoA mutase variant, IcmF, a member of this enzyme family that catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA also catalyzes the interconversion between isovaleryl-CoA and pivalyl-CoA, albeit with low efficiency and high susceptibility to inactivation. Given the biotechnological potential of the isovaleryl-CoA/pivalyl-CoA mutase (PCM) reaction, we initially attempted to engineer IcmF to be a more proficient PCM by targeting two active site residues predicted based on sequence alignments and crystal structures, to be key to substrate selectivity. Of the eight mutants tested, the F598A mutation was the most robust, resulting in an ∼17-fold increase in the catalytic efficiency of the PCM activity and a concomitant ∼240-fold decrease in the isobutyryl-CoA mutase activity compared with wild-type IcmF. Hence, mutation of a single residue in IcmF tuned substrate specificity yielding an ∼4000-fold increase in the specificity for an unnatural substrate. However, the F598A mutant was even more susceptible to inactivation than wild-type IcmF. To circumvent this limitation, we used bioinformatics analysis to identify an authentic PCM in genomic databases. Cloning and expression of the putative AdoCbl-dependent PCM with an α2β2 heterotetrameric organization similar to that of isobutyryl-CoA mutase and a recently characterized archaeal methylmalonyl-CoA mutase, allowed demonstration of its robust PCM activity. To simplify kinetic analysis and handling, a variant PCM-F was generated in which the αβ subunits were fused into a single polypeptide via a short 11-amino acid linker. The fusion protein, PCM-F, retained high PCM activity and like PCM, was resistant to inactivation. Neither PCM nor PCM-F displayed detectable isobutyryl-CoA mutase activity, demonstrating that PCM represents a novel 5'-deoxyadenosylcobalamin-dependent acyl-CoA mutase. The newly discovered PCM and the derivative PCM-F, have potential applications in bioremediation of pivalic acid found in sludge, in stereospecific synthesis of C5 carboxylic acids and alcohols, and in the production of potential commodity and specialty chemicals.

Keywords: adenosylcobalamin (AdoCbl); cofactor; enzyme; enzyme catalysis; enzyme kinetics; enzyme mutation; isomerase.

FIGURE 1.

Comparison of reactions catalyzed and organization of acyl-CoA mutases. A, reactions catalyzed by acyl-CoA mutases. IcmF can also catalyze the PCM reaction, albeit inefficiently. B, organization of ICM and PCM variants. ICM and PCM are heterotetramers in which two small B₁₂-binding subunits interact with two large substrate-binding subunits. IcmF is a natural variant in which the small and large subunits found in ICM are fused via a middle G-protein chaperone domain. PCM-F is an artificially engineered variant of PCM (this study) in which the large and small subunits have been fused via an 11-amino acid linker as described under “Experimental Procedures.”

FIGURE 2.

Substitutions at key active site residues underlie differences in substrate specificities in acyl-CoA mutases. A, multiple sequence alignment of substrate-binding domains of different AdoCbl-dependent acyl-CoA mutases. IcmF from C. metallidurans (YP_582365), PCM from X. autotrophicus strain Py2 (ABS70241), ICM from Streptomyces cinnamonensis (AAC08713), MCM from P. shermanii (YP_003687736), ECM from Rhodobacter sphaeroides (YP_354045), and HCM from Aquincola tertiaricarbonis strain L108 (AFK77668) are shown. Four residues (Phe-598, Gln-742, His-863, and Asn-901 in IcmF) predicted to be important for substrate binding, are highlighted in black. Identical and similar residues are indicated with asterisks and dots, respectively. Two residues, Phe and Gln, found in ICM and IcmF are substituted by Tyr and Arg in MCM and ECM or Ile and Gln in HCM, respectively. ECM differs from other acyl-CoA mutases by the substitution of His-863 and Asn-901 in IcmF to Gly and Pro, respectively. All accession numbers are from the NCBI Protein database. B, comparison of active site structures of IcmF (left) and MCM (right). The CmIcmF and P. shermanii MCM structures were generated using Protein Data Bank files 4XC6 and 4REQ, respectively. In the IcmF structure, n-butyryl-CoA is modeled in from Protein Data Bank file 4XC8. Two striking differences in the active site residues are the substitutions of Phe-598 and Gln-742 in IcmF (left) by Leu-87 and Asn-204 in PCM and Tyr-89 and Arg-207 in MCM (right), which are predicted to be important for substrate specificity. The histidine residues on the lower face of the corrin ring are provided by the protein and coordinate to the cobalt. In PCM, Leu-87 and Asn-204 are from the large subunit, whereas His-21 is from the small subunit.

FIGURE 3.

Enzyme-monitored turnover by wild-type and F598A IcmF. Spectra of (A) wild-type and (B) F598A IcmF (with 2 eq or 30 μm bound AdoCbl) in 50 mm HEPES-NaOH, pH 7.5, containing 100 mm NaCl and 10 mm MgCl₂ at 25 °C (solid lines) and after addition of 1 mm isovaleryl-CoA (dotted lines), which resulted in the formation of cob(II)alamin with an absorption maximum at 471 nm. C, rapid oxidation of cob(II)alamin to OH₂Cbl was observed during reaction of F598A IcmF (with 2 eq or 30 μm AdoCbl bound) with 1 mm isovaleryl-CoA in 50 mm HEPES-NaOH, pH 7.5, containing 100 mm NaCl and 10 mm MgCl₂ at 25 °C in the dark. D, the time-dependent increase at 351 nm (denoting OH₂Cbl formation) was plotted in the presence of oxygen. The data presented here are representative of at least three experiments and were fit to a single exponential equation described under “Experimental Procedures.”

FIGURE 4.

Effects of nucleotides on the isovaleryl-CoA mutase activity of IcmF. A and B, comparison of the time courses for the reaction catalyzed by (A) wild-type and (B) F598A IcmF at 37 °C in the absence or presence of nucleotides. The reaction conditions were 2 μm IcmF dimer, 100 μm AdoCbl, 50 mm HEPES-NaOH buffer, pH 7.5, 100 mm NaCl, 10 mm MgCl₂ ± 1 mm nucleotides, and 1 mm isovaleryl-CoA at 37 °C. The data represent the mean ± S.D. of three independent experiments. C and D, time dependence of the inactivation of wild-type (C) and F598A (D) IcmF in the absence or presence of nucleotides. The increase in absorbance at 351 nm corresponding to formation of OH₂Cbl via oxidation of the initially formed cob(II)alamin intermediate during turnover was monitored as described under “Experimental Procedures.” The spectra were recorded between 0 and 60 min. The data presented here are representative of at least three experiments and were fit to a single exponential equation described under “Experimental Procedures.”

FIGURE 5.

Characterization of PCM and PCM-F. A, the purity of the large and small subunits of PCM and PCM-F were judged by SDS-PAGE analysis. B, dependence of the isovaleryl-CoA mutase activity of PCM on the ratio of the small:large subunit. The reaction mixture contained 1 mm isovaleryl-CoA in 50 mm HEPES-NaOH buffer, pH 7.5, 100 mm NaCl, 10 mm MgCl₂ at 37 °C and varying ratios of the small:large subunit as described under “Experimental Procedures.” C, dependence of isovaleryl-CoA mutase activity of PCM (open circles) and PCM-F (closed circles) on the concentration of isovaleryl-CoA (0.1–1.0 mm) in 50 mm HEPES-NaOH buffer, pH 7.5, 100 mm NaCl, 10 mm MgCl₂ at 37 °C. The ratio of the small to large subunit of PCM was fixed at 3:1. The kinetic parameters derived from this analysis are reported in Table 1. D, time dependence of the inactivation of PCM (closed circles) and PCM-F (closed triangles). The inactivation of wild-type and F598A IcmF (Fig. 3D) are also included for comparison.

FIGURE 6.

PCM sequence comparison and gene organization. A, multiple sequence alignment of substrate-binding domains of PCMs. PCMs from X. autotrophicus (Xaut_5043), Burkholderia phenoliruptrix (locus tag BPACDRAFT_03564), Cycloclasticus sp. (locus tag Ga0055576_00391), and Nocardioides sp. CF8 (locus tag CF8_0950) are shown. The two conserved residues (Leu-87 and Asn-204, X. autotrophicus numbering) predicted to be important for substrate binding, are highlighted in black. Identical and similar residues are indicated with asterisks and dots, respectively. B, organization of the PCM-encoding operon from X. autotrophicus. The genes involved in the operon are annotated as paaK (phenylacetate-CoA ligase-like, Xaut_5040), paaD (phenylacetic acid degradation protein-like, Xaut_5041), a meaB homolog encoding a G-protein chaperone (Xaut_5042), pcmA (large subunit of PCM, Xaut_5043), pcmB (small subunit of PCM, Xaut_5044), and tetR (a transcriptional regulator, Xaut_5045).