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

Background: 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. Conclusion: A B12-dependent PCM was identified, cloned, and expressed and exhibited PCM activity. Significance: The newly discovered PCM could be useful in bioremediation and biosynthetic reactions. 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.

GTPase activity and AdoCbl-dependent isomerase activity. Recently, 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). The structural difference between isobutyryl-CoA and the substrate analog, isovaleryl-CoA, is but one carbon unit. From a biotechnological viewpoint, IcmF could be potentially useful as a reagent for bioremediation of pivalic acid found in pharmaceutical wastewaters (pivalic acid esters are used as pro-drugs) and/or for the biosynthesis of a simple ␤-nonfunctional ␣-quaternary carboxylic acid (3,14). Another potential application of IcmF is in metabolic engineering of pathways to produce branched C4 and C5 building blocks that can subsequently be converted to useful derivatives, for example, the corresponding isobutyl and pivalyl alcohols. The potential utility of CmIcmF for isomerizing isovaleryl-CoA/pivalyl-CoA is, however, limited by its ineffi-ciency; it displays an ϳ1000-fold lower catalytic efficiency for the isomerization of isovaleryl-CoA versus isobutyryl-CoA and a high susceptibility to inactivation (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). The residues corresponding to Phe-598 and Gln-742 in CmIcmF are Tyr-89 and Arg-207 in the Propionibacterium shermanii MCM, where they interact with the carboxylate moiety of the substrate (Fig. 2B). In HCM, the corresponding amino acids are Ile and Gln. Although the substitution of Arg in MCM by Gln in IcmF, ICM, and HCM likely reflects the absence of an anionic carboxylate substituent in their respective substrates, the substitution of Tyr-89 (in MCM) by Phe (in IcmF/ICM) and Ile in (HCM) could be important for accommodating differences in substrate bulk (12,13). Tyr-89 in MCM is also proposed to play a role in labializing the cobalt-carbon bond of AdoCbl following substrate binding (18). Although the residues corresponding to Tyr-89 and Arg-207 are also found in ECM, substitutions at two other active site residues are predicted to be important for its substrate specificity. ECM catalyzes the interconversion of ethylmalonyl-CoA and methylsuccinyl-CoA, which are one-carbon longer than the corresponding substrates for MCM (10). In fact, MCM can also catalyze the successive isomerization of glutaryl-CoA to methylsuccinyl-CoA and ethylmalonyl-CoA, albeit with very low efficiency (19). Substitutions of His-328 and Asn-366 in MCM by Gly and Pro are proposed to accommodate the longer substrate in ECM. The MCM residues Tyr-89 and Arg-207 are conserved in ECM and presumably make similar contacts with the carboxylate substituent of the substrate as they do in MCM (Fig. 2).
In this study, we engineered CmIcmF to enhance its isovaleryl-CoA mutase activity at the expense of its isobutyryl-CoA mutase activity by introducing mutations at Phe-598 and Gln-742 (Fig. 2B). The F598A mutation switched the substrate specificity of IcmF and enhanced the catalytic efficiency of the isovaleryl-CoA mutase over the native isobutyryl-CoA mutase activity ϳ4000-fold. However, the mutation also 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 herein, we report its initial characterization.

Experimental Procedures
Materials-AdoCbl, GTP, GDP, isobutyryl-CoA, isovaleryl-CoA, and DL-3-hydroxybutyryl-CoA were purchased from Sigma. Valeric acid was purchased from Fluka. Dithiothreitol and tris(2-carboxyethyl)phosphine hydrochloride were from Gold Biotechnology (St. Louis, MO). All other chemicals were purchased from Sigma or Fisher Scientific, and were used without further purification.
Site-directed Mutagenesis of CmIcmF-Mutagenesis was performed using the QuikChange XL site-directed mutagenesis kit (Agilent Technologies) using the CmIcmF cloned in the pMCSG7 plasmid as a template. The following set of forward primers in which the mutagenic codon is underlined were syn-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 B 12 -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." thesized by Integrated DNA Technologies (Coralville, IA) and  used to introduce missense mutations: F598A, 5Ј-CCC ACG  CGC ATG GCC GCG GGC GAG GG-3Ј; F598G, 5Ј-CCC ACG  CGC ATG GGC GCG GGC GAG GG-3Ј; F598I, 5Ј-CC ACG  CGC ATG ATC GCG GGC GAG-3Ј; F598L, 5Ј-CCC ACG  CGC ATG TTA GCG GGC GAG GGC GAT G-3Ј; F598V,  5Ј-CC ACG CGC ATG GTC GCG GGC GAG-3Ј; Q742A,  5Ј-CTG AAG GAA GAC CAG GGC GCG AAT ACG TGC  ATC TTC TC-3Ј; Q742L, 5Ј-AAG GAA GAC CAG GGC CTG  AAT ACG TGC ATC TTC TC-3Ј; Q742N, 5Ј-CTG AAG GAA  GAC CAG GGC AAT AAT ACG TGC ATC TTC TCG-3Ј. The sequences of the reverse primers were complementary to those of the forward primers. The mutations were confirmed by nucleotide sequence determination at the University of Michigan DNA Sequencing Core.
Expression and Purification of CmIcmF-Recombinant wildtype and mutant IcmFs were expressed and purified as described previously (13).
Cloning of PCM Subunits-The genomic DNA from X. autotrophicus strain Py2 (ATCC BAA-1158) was purchased from ATCC (Manassas, VA). The large and small subunits of PCM cDNAs were PCR-amplified by PfuTurbo High-Fidelity DNA polymerase (Agilent Technologies) using the following primers containing NdeI and HindIII restriction sites (underlined): large subunit (forward): 5Ј-AGT ATA CAT ATG AAC CAG GCC GCG GTG-3Ј, and large subunit (reverse), 5Ј-T AGT AAG CTT TCA GGC GAT GAT TAG GGG ATG-3Ј; small subunit (forward), 5Ј-CTA GTA CAT ATG ATC CAT GCC GGC ACG-3Ј, and small subunit (reverse), 5Ј-A GAT AAG CTT TCA TGG GTT TGC CTC CG-3Ј. The PCR products of the large and small subunits were digested with NdeI and HindIII and subcloned into the pET-28 vector (Novagen).
Subsequently, the resulting vector was used as a template for ligation-independent cloning (LIC) for the large subunit with the following primers, 5Ј-TAC TTC CAA TCC AAT GCA  ATG AAC CAG GCC GCG GTG-3Ј (forward) and 5Ј-T TAT  CCA CTT CCA ATG TTA TTA TCA GGC GAT GAT TAG  GGG ATG-3Ј (reverse), and subcloned into a LIC vector, which introduces a His 6 -MBP tag and tobacco etch virus (TEV) protease cleavage site at the N terminus of the desired protein.
Three additional residues, Ser-Asn-Ala, remained at the N terminus of the large subunit of PCM after removal of the His 6 -MBP tag.
Construction of an Expression Plasmid for Fusion Protein between Large and Small PCM Subunits-To generate a fusion of the large and small subunits of PCM (PCM-F), a co-expression system was initially constructed using pETDuet-1 vector (Novagen), in which the large subunit was inserted into multicloning site 1 (MCS1) using XbaI and HindIII restriction sites, and the small subunit was inserted into MCS2 using NdeI and XhoI restriction sites. Subsequently, the second translation initiation site between MCS1 and MCS2 was deleted and an 11amino acid linker sequence, (Gly-Gln) 5 -Gly, was inserted between the large and small subunits simultaneously by the inverse PCR method using the following 5Ј-phosphorylated primers: 5Ј-GGA CAA GGA CAA GGA CAA GGA CAA GGA CAA GGA ATG ATC CAT GCC GGC ACG-3Ј (forward) and 5Ј-GGC GAT GAT TAG GGG ATG GC-3Ј (reverse). The resulting fragment was ligated and transformed, and subsequently used as a template for LIC. The insert was amplified with the following primers for LIC cloning, 5Ј-TAC TTC CAA TCC AAT GCA ATG AAC CAG GCC GCG GTG-3Ј (forward) and 5Ј-T TAT CCA CTT CCA ATG TTA TTA TCA TGG GTT TGC CTC CGA-3Ј (reverse), and inserted into a LIC vector, which introduces a His 6 -SUMO tag and TEV protease cleavage site at the N terminus of the desired protein. Three residues, Ser-Asn-Ala, were added to the N terminus of the expressed protein following removal of the His 6 -SUMO tag.
Expression and Purification of PCM and PCM-F-Escherichia coli BL21(DE3) was transformed with the vector expressing the large subunit of PCM and grown overnight at 37°C in 5 ml of Luria-Bertani medium containing ampicillin (100 g/ml). Then, 6 liters of the same medium containing ampicillin was inoculated with the starter culture and grown at 37°C. After 4 h, when the A 600 had reached 0.6 -0.8, the temperature was reduced to 15°C. The cultures were induced with 0.1 mM isopropyl ␤-D-1-thiogalactopyranoside, and the cells were harvested 20 h later. The cells pellets were stored at Ϫ80°C until use. The cell pellets were suspended in 150 ml of buffer containing 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 20 mM imidazole, 0.2 mg/ml of lysozyme, 1 mM PMSF and one protease inhibitor mixture tablet (Roche Applied Science). The cell suspension was stirred at 4°C for 40 min and then sonicated (power setting ϭ 6) on ice for 10 min at 30-s intervals separated by 60-s cooling periods. The sonicate was centrifuged at 35,000 ϫ g for 30 min, and the supernatant was loaded onto a Ni-Sepharose 6 Fast Flow column (2.5 ϫ 8 cm, GE Healthcare) pre-equilibrated with Buffer A (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 20 mM imidazole). The column was washed with 300 ml of Buffer A containing 20 mM imidazole and eluted with 300 ml of a linear gradient ranging from 20 to 300 mM imidazole in Buffer A. The fractions of interest were identified by SDS-PAGE analysis, pooled, and concentrated to 5 ml, and dialyzed overnight against 1 liter of dialysis buffer containing 100 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 1 mM 2-mercaptoethanol and His 6 -MBP tag was cleaved with TEV protease (1 mg of TEV protease/100 mg of protein). The dialyzed protein was loaded on to amylose resin column (New England Biolabs) to remove His 6 -MBP tag, and the flow-through fraction was collected and loaded on to a Superdex 200 column (120 ml, GE Healthcare) pre-equilibrated with 50 mM HEPES-NaOH, pH 7.5, 100 mM NaCl. The fractions of interest were pooled, concentrated, frozen in liquid nitrogen, and stored at Ϫ80°C until further use. The His 6 -tagged small subunit of PCM was expressed as described for the large subunit with the exception that kanamycin (50 g/ml) was used as the antibiotic and purified using a Ni-Sepharose 6 Fast Flow column and followed by gel filtration using a Superdex 200 column. PCM-F was expressed as a fusion with a His 6 -tagged SUMO protein and expressed and purified using the same procedures as described above for the large subunit with the exception of the amylose resin column, which was excluded.
Enzyme Assays-Enzyme activity was monitored in 50 mM HEPES-NaOH, pH 7.5, containing 100 mM NaCl and 10 mM MgCl 2 in a total volume of 0.6 ml. The reaction mixture contained 0.5-20 M enzyme, 100 M AdoCbl, and 0.1-5 mM isovaleryl-CoA or isobutyryl-CoA Ϯ 1 mM nucleotides (GTP or GDP) at 37°C. The enzymes were preincubated with AdoCbl (Ϯ 1 mM nucleotides in the case of IcmF), and the reaction was started by addition of substrate. At various time points (0.5-60 min), 100-l aliquots were removed and quenched with 50 l of 2 N KOH containing 0.2 mM valeric acid used as an internal standard. Following addition of 50 l of H 2 SO 4 (15%, v/v), the reaction mixture was saturated with solid NaCl and extracted with ethyl acetate (125 l). The extract was analyzed directly by gas chromatography (GC, Agilent Technologies) using a 0.25-m DB-FFAP (30-m ϫ 0.25 mm inner diameter) capillary column (Agilent Technologies). A 5-l sample was injected in the pulsed splitless mode. The oven temperature was initially at 80°C. Following the sample injection, the temperature was raised to 150°C at a rate of 10°C/min and maintained at 150°C for 2 min. Retention times for the compounds of interest were as follows: isobutyric acid, 5.85 min; pivalic acid, 5.99 min; n-butyric acid, 6.5 min; isovaleric acid, 6.96 min; and valeric acid, 7.78 min.
To determine the kinetic parameters for the isovaleryl-CoA mutase and isobutyryl-CoA mutase activities of IcmF, the initial rate of the reaction was plotted against substrate concentration and fitted to the Michaelis-Menten equation To determine the kinetic parameters of isovaleryl-CoA mutase activity of PCM, the initial rate of the reaction was plotted against substrate concentration and fitted to the Hill equa- Enzyme-monitored Turnover-Spectral changes in AdoCbl bound to IcmF and PCM were monitored by UV-visible spectroscopy at 25°C in 50 mM HEPES-NaOH, pH 7.5, containing 100 mM NaCl and 10 mM MgCl 2 . Substrates (1 mM final concentration of isovaleryl-CoA or isobutyryl-CoA) were added to 30 M AdoCbl-loaded enzyme. With IcmF, the influence of the G-protein domain on catalytic turnover was checked by addition of 5 mM GTP or GDP to the reaction mixture. The amount of cob(II)alamin formed under steady-state turnover conditions was calculated from the decrease in absorbance at 525 nm upon substrate addition using a value of ⌬⑀ 525 nm of Ϫ4.8 mM Ϫ1 cm Ϫ1 (20). During the course of the reaction, AdoCbl was gradually converted to enzyme-bound aquocobalamin (OH 2 Cbl) as indicated by the appearance of the 351-nm absorption peak. The increase in absorption at 351 nm (A) was fitted to a single exponential function, where k is the observed rate constant for inactivation, A 0 is the initial absorbance at 351 nm, t is time in minutes, and A 1 is the amplitude.
Isothermal Titration Calorimetry-Isothermal titration calorimetry experiments were performed in triplicate as described previously (12) and the data were analyzed using Microcal ORIGIN software. Briefly, binding of AdoCbl to PCM was monitored as follows. Enzyme (10 M small subunit of PCM or PCM-F) in 50 mM HEPES-NaOH buffer, pH 7.5, containing 100 mM NaCl was titrated with 30 ϫ 10-l aliquots of a 0.4 mM solution of AdoCbl at 20.0°C. The calorimetric signals were integrated and the data were well fitted to a single-site binding model to estimate the equilibrium association constant, K A , and the binding enthalpy, ⌬H 0 . The Gibbs free energy of binding, ⌬G 0 , and the entropic contribution to the binding free energy, ϪT⌬S 0 , were calculated using Equations 1 and 2.
Bioinformatics Analysis-The integrated microbial genomes database web-based tools were used for BLAST search and operon analysis (21). A multiple sequence alignments were constructed using a stand-alone version of ClustalX version 2.1.

Results
Structure-based Redesign of IcmF-Our efforts at enhancing the catalytic activity of IcmF with isovaleryl-CoA as substrate were guided by sequence comparisons of IcmF with other acyl-CoA mutases (10,11) and by the crystal structures of IcmF (17) and MCM (16,22). Sequence comparisons revealed the presence of a putative acyl-CoA mutase annotated as MCM-like in which Leu and Asn substitute Phe-598 and Gln-742, respectively, in IcmF ( Fig. 2A). Hence, these two residues were targeted by multiple missense mutations (Table 1). To better accommodate the longer isovaleryl-CoA substrate, we introduced residues with smaller side chains at Phe-598 (Ala, Gly, Ile, Leu, and Val) and Gln-742 (Ala, Leu, and Asn). The yield of each of the purified mutants (ϳ5.0 mg/liter of culture) was similar to that of wild-type IcmF and the purity of each mutant protein was judged to be Ͼ95% by SDS-PAGE analysis (data not shown). The mutants, like wild-type IcmF, eluted as a single peak by gel filtration chromatography with a molecular mass of 279 kDa, consistent with being homodimers.
Isovaleryl-CoA Mutase and Isobutyryl-CoA Mutase Activities of IcmF-The isovaleryl-CoA mutase and isobutyryl-CoA mutase activities of the mutants were compared with wild-type IcmF ( Table 1). The F598A mutation simultaneously enhanced isovaleryl-CoA mutase activity 6-fold (0.18 Ϯ 0.02 mol min Ϫ1 mg Ϫ1 ) and diminished isobutyryl-CoA activity Ͼ600-fold (0.039 Ϯ 0.003 mol min Ϫ1 mg Ϫ1 ), with respect to wild-type IcmF (0.030 Ϯ 0.001 mol min Ϫ1 mg Ϫ1 and 24 Ϯ 3 mol min Ϫ1 mg Ϫ1 , respectively). Substitution of Phe-598 with the nonaromatic hydrophobic residues isoleucine, leucine, and valine, either maintained or increased isovaleryl-CoA mutase activity, whereas isobutyryl-CoA mutase activity was diminished albeit with less dramatic effects than the F598A mutation. In addition, mutations at Phe-598 decreased the K m for isovaleryl-CoA and therefore increased the catalytic efficiency (k cat /K m ) of the isovaleryl-CoA mutase reaction compared with wild-type IcmF. The activity of the F598G mutant was not detectable with either substrate. Mutation of Gln-742 to alanine, leucine, or asparagine, resulted in undetectable isovaleryl-CoA mutase activity and significantly diminished isobutyryl-CoA mutase activity (Table 1).
Inactivation Kinetics of IcmF Mutants-The active sites in the CmIcmF dimer exhibit identical affinity for AdoCbl (K d ϭ 0.27 Ϯ 0.01 M, n ϭ 2.1 Ϯ 0.1) in contrast to the different affinities reported for the Geobacillus kaustophilus IcmF (K d1 ϭ 0.081 Ϯ 0.014 M and K d2 ϭ 1.98 Ϯ 0.42 M) (12). To assess whether the low or undetectable activity of several of the CmIcmF mutants in Table 1 was due to their enhanced propen-   sity for inactivation, the spectrum of AdoCbl bound to IcmF was monitored following addition of isovaleryl-CoA (Fig. 3).
For these experiments, IcmF was reconstituted with 2 eq of AdoCbl and the absence of free AdoCbl was confirmed by analyzing the spectrum of the filtrate obtained after concentrating the sample mixture using an Amicon concentrator (50-kDa cutoff). Addition of isovaleryl-CoA to wild-type holo-IcmF resulted in formation of the intermediate, cob(II)alamin, as evidenced by the decrease in absorbance at 530 nm and increase at 471 nm (Fig. 3A). Using a ⌬⑀ 525 nm of Ϫ4.8 mM Ϫ1 cm Ϫ1 (20), cob(II)alamin was estimated to represent ϳ20% of the cofactor under steady-state turnover conditions (Table 2). In contrast, AdoCbl was quantitatively converted to cob(II)alamin when isovaleryl-CoA was added to F598A (Fig. 3B) or to the F598I/L or Q742A mutants ( Table 2). Accumulation of all the cofactor as cob(II)alamin indicates a change in the reaction coordinate such that the barrier to one or more steps following cob(II)alamin formation and preceding reformation of AdoCbl, has increased.
Because cob(II)alamin can be oxidized to OH 2 Cbl, its increased accumulation in the mutants could lead to an increased propensity for OH 2 Cbl formation. OH 2 Cbl is a hallmark of inactivation in AdoCbl-dependent enzymes (23) and is characterized by an increase in absorption at 351 and 530 nm (Fig. 3C). The F598A mutant inactivates 3-fold more rapidly than wild-type IcmF as monitored by the increase in absorbance at 351 nm (Fig. 3D, Table 2).
The F598G and Q742N mutants showed rapid conversion of AdoCbl to OH 2 Cbl under aerobic conditions without detectable accumulation of the cob(II)alamin intermediate (Table 2). Hence, enzyme-monitored turnover indicates that the IcmF mutants engineered to have enhanced isovaleryl-CoA mutase activity are simultaneously more susceptible to inactivation. The only exception is Q742L IcmF, which showed a lower proportion of the cofactor in the cob(II)alamin state under steady-  state turnover conditions and a lower rate of OH 2 Cbl formation ( Table 2). However, Q742L IcmF did not exhibit isovaleryl-CoA mutase activity ( Table 1).

Effects of Nucleotides and Reductants on the Isovaleryl-CoA
Mutase Activity of IcmF-Next, the ability of nucleotides that bind to the G-protein domain of IcmF, on isovaleryl-CoA mutase activity was assessed with the most active mutant, F598A. The G-protein domain interacts with both the B 12 and substratebinding domains in IcmF (17). The presence of GTP or GDP increased the maximal isovaleryl-CoA mutase activity of wild-type IcmF by ϳ4-fold (Fig. 4A). However, the same conditions had virtually no effect on the isovaleryl-CoA mutase activity of the F598A mutant (Fig. 4B). Furthermore, whereas the nucleotides stabilized wild-type IcmF against inactivation, they had little or no effect on the F598A mutant (Fig. 4, C and D).
In a further attempt to stabilize IcmF against inactivation, the effect of several reductants was tested. Both DTT and 2-mercaptoethanol enhanced the maximal isovaleryl-CoA mutase activity of wild-type IcmF ϳ5-fold and F598A IcmF ϳ2-fold, respectively, whereas GSH and tris(2-carboxyethyl)phosphine hydrochloride were without effect (data not shown).
Identification of a Putative Pivalyl-CoA/Isovaleryl-CoA Mutase-Given the high susceptibility of the most active IcmF mutant to inactivation, we used bioinformatics analysis to identify a "bona fide" isovaleryl-CoA/pivalyl-CoA mutase. We examined sequences that are annotated in the databases as MCM-like proteins but differ in the two active site residues that are predicted to be important for substrate specificity especially in organisms with multiple copies of acyl-CoA mutases (24,25). Specifically, we identified a group of genes that encode a substrate-binding subunit where Leu and Asn substitute Phe-598 and Gln-742, respectively, in IcmF. We concentrated on X. autotrophicus strain Py2 where Xaut_5043 and Xaut_5044 encode the large and small subunits, respectively, of an AdoCbl-dependent mutase, tentatively identified as a PCM. In the large subunit of the putative PCM, Leu-87 and Asn-204 correspond to Phe-598 and Gln-742 in IcmF, respectively (Fig. 2). In addition, a gene encoding a putative G-protein chaperone (Xaut_5042) is present in the same operon. The bacterial MeaB and human CblA are G-protein chaperones that gate docking of AdoCbl to bacterial and human MCM, respectively, whereas the corresponding G-protein in IcmF is fused between the small and large subunits (Fig. 1B). The putative PCM is similar in organization to ICM, which exists as an ␣ 2 ␤ 2 heterotetramer with the small and large subunits binding AdoCbl and substrate, respectively (9).
Purification and Kinetic Properties of PCM-The recombinant large subunit of PCM was purified as a His 6 -MBP-tagged protein. Following cleavage of the tag, the large subunit was obtained with a yield of ϳ4 mg of protein/liter of culture. The band observed by SDS-PAGE analysis corresponds to the predicted mass of 62.3 kDa for the large subunit (Fig. 5A). Size exclusion chromatography on a calibrated Superdex 200 column yielded an estimated molecular mass of 135 kDa, consistent with the large subunit being a homodimer (not shown). The recombinant small subunit of PCM was purified as an N-terminal His 6 -tagged protein and ϳ6 mg of highly pure protein were obtained per liter of culture (Fig. 5A). The small subunit eluted with an apparent mass of 20 kDa by gel filtration chromatography, suggesting that it exists as a monomer based on the predicted mass of the polypeptide of 17.7 kDa. Gel filtration of a 1:1 mixture of the large and small subunits of PCM in the presence of AdoCbl, showed no evidence of complex formation, indicating weak interaction between the subunits under these conditions. Next, the dependence of the isovaleryl-CoA mutase activity of PCM was assessed at increasing small:large subunit ratios (Fig. 5B). Maximal activity was observed at an ϳ1:1 ratio. The specific activity (0.18 Ϯ 0.04 mol min Ϫ1 mg Ϫ1 of protein) and steady-state kinetic parameters for PCM were determined at a 3:1 molar ratio of the small:large subunit. The activity of PCM showed a sigmoidal dependence on the concentration of isolvaleryl-CoA (Fig. 5C). From a Hill plot analysis of the data, the following values were estimated: K m for isovaleryl-CoA ϭ 0.37 Ϯ 0.13 mM, k cat ϭ 14 Ϯ 3 min Ϫ1 , and n (Hill coefficient) ϭ 1.4 Ϯ 0.4, indicating positive cooperativity. Unlike IcmF, the activity of PCM was resistant to inactivation (Table 2 and Fig. 5D).
Purification and Kinetic Characteristics of PCM-F-Because the weak interaction between the large and small subunits of PCM complicates kinetic analysis, we created a variant in which the large and small subunits were linked in a single polypeptide by an 11-amino acid long (Gly-Gln) 5 -Gly sequence to give the fusion protein, PCM-F (Fig. 1B). This strategy has been used previously to study an AdoCbl-dependent isomerase, glutamate mutase, which like PCM, exhibits an ␣ 2 ␤ 2 , stoichiometry (26). Highly pure PCM-F was obtained at a yield of ϳ2 mg of protein/ liter of culture (Fig. 5A). Gel filtration chromatography yielded an apparent mass of 168 kDa indicating that PCM-F with a subunit molecular mass of 78.8 kDa, exists as an ␣ 2 homodimer.
The activity of PCM-F showed a sigmoidal dependence on the concentration of isovaleryl-CoA (Fig. 5C). From a Hill plot analysis of the data, the following values were estimated: K m for isovaleryl-CoA ϭ 0.20 Ϯ 0.01 mM, k cat ϭ 23 Ϯ 1 min Ϫ1 , and n (Hill coefficient) ϭ 2.0 Ϯ 0.3. The catalytic efficiency (k cat /K m ) of PCM-F is 3-fold higher than for PCM.
Binding of AdoCbl to PCM and PCM-F-The energetics of AdoCbl binding to the PCM and PCM-F were monitored by isothermal titration calorimetry ( Table 3). Binding of AdoCbl to the small subunit of PCM (K d ϭ 12.5 Ϯ 1.9 M) exhibits a ⌬G of Ϫ6.6 Ϯ 0.1 kcal/mol and is enthalpically favored. The K d for AdoCbl is lower for PCM-F (4.9 Ϯ 0.3 M) than for PCM and exhibits a ⌬G of Ϫ7.3 Ϯ 0.2 kcal/mol, which is also enthalpically driven. Hence, the presence of the "built-in" large subunit in PCM-F enhances the affinity for AdoCbl ϳ2.6-fold.

Discussion
Our understanding of pivalic acid metabolism in nature is very limited. Some bacteria are known to mineralize pivalic acid to carbon dioxide (14). Some bacteria use pivalic acid as a  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. starter unit and incorporate it into tertiary-butyl fatty acids, iso-even or anteiso-fatty acids and in Streptomyces avermitilis, into the antibiotic avermectin (27). In addition, pivalic acid (or pivalyl-CoA) might be formed during catabolism of marine sponge-derived polytheonamides in which the tert-butyl modification of threonine is introduced post-translationally (28). Isovaleryl-CoA is produced during leucine catabolism via the branched chain keto acid dehydrogenase or in myxobateria, via a mevalonate-dependent isoprenoid biosynthesis pathway involving 3-hydroxy-3-methylglutaryl-CoA synthase (29). In several bacteria, the gene encoding IcmF is coregulated with those involved in branched-chain amino acid degradation (30) or fatty acid catabolism (13). The potential for utilizing a catalyst with isovaleryl-CoA/pivalyl-CoA isomerase activity for metabolic engineering to produce branched C4 and C5 building blocks or the corresponding alcohol derivatives, is untapped.
Until this study, IcmF was the only known albeit inefficient, catalyst with isovaleryl-CoA/pivalyl-CoA isomerase activity, and with high susceptibility to inactivation (13). The PCM described in this study is derived from X. autotrophicus strain Py2, a facultative aerobe that was first isolated from black pool sludge, has versatile metabolic capabilities, and is able to utilize 2-hydroxyisobutyric acid, propene, and trichloroethylene, 1-butene among others, as carbon sources (31). The large subunit of PCM has Leu and Asn residues that are also seen in other genes annotated as being MCM-like in the database (Fig. 6A). At this point, it is not clear whether or not these AdoCbl-dependent acyl-CoA mutases, which are distinct from all previously characterized ones, are indeed bona fide PCMs. In X. autotrophicus strain Py2, the PCM genes are located in an operon that also encodes a homolog of the G-protein chaperone described for MCM, and two other genes annotated as phenylacetic acid degradation protein-like (paaD) and phenylacetic acid-CoA ligase-like (paaK) (Fig. 6B). Although the ligase is presumably important for converting the acid to a CoA ester needed for the AdoCbl-dependent isomerase activity, the role of PCM in X. autotrophicus remains to be established. In E. coli, 14 genes are involved in the phenylacetic acid degradation pathway (32).
Most acyl-CoA mutases exhibit fairly stringent substrate specificities, which appear to be dictated by a limited number of differences in key active site residues. In an effort to exploit the measurable but low isovaleryl/pivalyl-CoA mutase activity reported for wild-type IcmF (13), we rationally targeted two active site residues, Phe-598 and Gln-742, making a total of eight substitutions. Of the individual missense mutations, F598A IcmF was the most active and led to an ϳ4000-fold enhancement of the isovaleryl-CoA mutase relative to the isobutyryl-CoA mutase activity compared with wild-type enzyme (Table 1). However, F598A IcmF and the other mutants were even more susceptible than wild-type IcmF to inactivation during turnover (Table 2), a problem that was only marginally improved by the presence of reductants, DTT and 2-mercaptoethanol.
A similar switch in substrate selectivity has been previously reported for MCM where the corresponding active site residues, Tyr-89 and Arg-207, were targeted by mutagenesis (33). The double mutant, Y89F/R207Q, mimicked the corresponding active site residues in ICM (and the then unknown IcmF). In contrast to wild-type MCM, the double mutant bound the ICM substrates, n-butyryl-CoA and isobutyryl-CoA, but instead of catalyzing their isomerization, promoted inactivation via a suicidal internal electron transfer mechanism. Among the mutants characterized in this study, the F598I IcmF resembles HCM with Ile-598 and Gln-742 corresponding to Ile-90 and Gln-208 in HCM (11). Therefore, we examined the possibility that F598I IcmF exhibits HCM activity. However, DL-3-hydroxybutyryl-CoA was not converted to product as judged by an HPLC-based assay (data not shown) although cob(II)alamin formation was seen indicating that the first step, cobalt-carbon bond homolysis, had occurred. These results indicate that residues that dictate the narrow substrate specificity of acyl-CoA mutases are distinct from those that promote the 1,2 rearrangement reactions. Alternatively, mutations that permit accom- 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).

TABLE 3 Thermodynamic parameters for the binding of AdoCbl to PCM
The experiments were performed in 50 mM HEPES-NaOH, pH 7.5, 100 mM NaCl at 20°C as described under "Experimental Procedures." The data represent the mean Ϯ S.D. of three independent experiments. modation of non-native substrates also lead to a misalignment in the active site residues so that unwanted side reactions such as oxidation of cob(II)alamin, are suppressed. Loss of reaction fidelity is seen in mutants in other AdoCbl-dependent enzymes, e.g. diol dehydratase, which catalyzes the conversion of 1,2propanediol and 1,2-ethanediol to the corresponding aldehydes, and undergoes mechanism-based inactivation in the presence of glycerol. Interestingly, the S301A and Q336A mutations in the catalytic subunit of diol dehydratase confer resistance to glycerol-dependent inactivation relative to the wild-type enzyme indicating that these residues are involved in inactivation in the native enzyme (34). The susceptibility of AdoCbl-dependent radical enzymes to irreversible inactivation during catalysis necessitates their reliance on chaperones for their reactivation. In the subclass of AdoCbl-dependent enzymes to which diol dehydratase belongs, reactivating factors mediate an ATP-dependent exchange of enzyme-bound OH 2 Cbl with free AdoCbl (23,35). These reactivases have sequence similarity to DnaK and other members of the Hsp70 family of molecular chaperones and lower sequence similarity to the large subunits of corresponding mutases (23). In contrast, the G-protein chaperones associated with AdoCbl-dependent acyl-CoA mutases belong to the G3E family of P-loop metallochaperones (36). In MCM where the role of the G-protein chaperone MeaB is best characterized (37), the chaperone uses GTP hydrolysis to power expulsion of cob(II)alamin when 5Ј-deoxyadenosine is lost from the active site (37). MeaB diminishes the inactivation rate of the mutase ϳ15and ϳ3-fold in the presence of GTP and GDP, respectively (20). The human ortholog of MeaB is CblA and it is reported to prevent inactivation and increase the enzymatic activity of human MCM (38,39). In IcmF, which has a built-in G-protein chaperone, GTP and GDP protect against inactivation in wild-type but not in F598A IcmF (Fig. 4, Table 2) indicating that this residue might be important for communication between the mutase and G-protein domains. The GTPase activity of IcmF is not affected in the Phe-598 mutants (k cat is 30 -40 min Ϫ1 ), which is comparable with that of wild-type IcmF (34 Ϯ 2 min Ϫ1 at 37°C).
In summary, our efforts to rationally redesign the IcmF active site to improve its isovaleryl-CoA mutase activity yielded a protein that was a better catalyst but also more susceptible to inactivation. They also led to characterization of the first AdoCbldependent PCM exhibiting isovaleryl-CoA mutase activity, which was resistant to inactivation. We engineered a variant in which the two weakly interacting subunits of PCM were fused into a single polypeptide to obtain PCM-F, which was also highly active as an isovaleryl-CoA mutase. PCM-F could be useful in applications ranging from bioremediation to stereospecific synthesis of C5 carboxylic acids and alcohols.