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.
Highlights
IcmF exhibits low isovaleryl-CoA/pivalyl-CoA mutase (PCM) activity
Sequence alignment of the active site residues in acyl-CoA mutases indicate substitutions at a few key positions that are predicted to be important for substrate specificity (Fig. 2A) [11,12,13, 15], which can be located in the crystal structures of methylmalonyl-CoA mutase (MCM) [16] and CmIcmF [17] (Fig. 2B)
The mutation enhanced the propensity of the enzyme to inactivation during turnover. This prompted us to use bioinformatics analysis to identify a PCM from Xanthobacter autotrophicus and we report its initial characterization
Summary
IcmF exhibits low isovaleryl-CoA/pivalyl-CoA mutase (PCM) activity. Results: IcmF mutants designed to enhance PCM activity were susceptible to inactivation prompting a bioinformatics search for a “bona fide” PCM. 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. We demonstrated that the Cupriavidus metallidurans (Cm) IcmF catalyzes an additional reaction, i.e. isomerization of isovaleryl-CoA and pivalyl-CoA, designated as pivalyl-CoA mutase (PCM) activity (Fig. 1A) [13]. Sequence alignment of the active site residues in acyl-CoA mutases indicate substitutions at a few key positions that are predicted to be important for substrate specificity (Fig. 2A) [11,12,13, 15], which can be located in the crystal structures of MCM [16] and CmIcmF [17] (Fig. 2B). This prompted us to use bioinformatics analysis to identify a PCM from Xanthobacter autotrophicus and we report its initial characterization
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