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Coupling of hydrogenic tunneling to active-site motion in the hydrogen radical transfer catalyzed by a coenzyme B12-dependent mutase.

ABSTRACT: Hydrogen transfer reactions catalyzed by coenzyme B(12)-dependent methylmalonyl-CoA mutase have very large kinetic isotope effects, indicating that they proceed by a highly quantal tunneling mechanism. We explain the kinetic isotope effect by using a combined quantum mechanical/molecular mechanical potential and semiclassical quantum dynamics calculations. Multidimensional tunneling increases the magnitude of the calculated intrinsic hydrogen kinetic isotope effect by a factor of 3.6 from 14 to 51, in excellent agreement with experimental results. These calculations confirm that tunneling contributions can be large enough to explain even a kinetic isotope effect >50, not because the barrier is unusually thin but because corner-cutting tunneling decreases the distance over which the system tunnels without a comparable increase in either the effective potential barrier or the effective mass for tunneling.

SUBMITTER: Dybala-Defratyka A

PROVIDER: S-EPMC1904141 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Coupling of hydrogenic tunneling to active-site motion in the hydrogen radical transfer catalyzed by a coenzyme B12-dependent mutase.

Dybala-Defratyka Agnieszka A Paneth Piotr P Banerjee Ruma R Truhlar Donald G DG

Proceedings of the National Academy of Sciences of the United States of America 20070620 26

Hydrogen transfer reactions catalyzed by coenzyme B(12)-dependent methylmalonyl-CoA mutase have very large kinetic isotope effects, indicating that they proceed by a highly quantal tunneling mechanism. We explain the kinetic isotope effect by using a combined quantum mechanical/molecular mechanical potential and semiclassical quantum dynamics calculations. Multidimensional tunneling increases the magnitude of the calculated intrinsic hydrogen kinetic isotope effect by a factor of 3.6 from 14 to ...[more]

PMID: 17581872

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Project description: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.

Project description:The ratio of the major monensin analogs produced by Streptomyces cinnamonensis is dependent upon the relative levels of the biosynthetic precursors methylmalonyl-coenzyme A (CoA) (monensin A and monensin B) and ethylmalonyl-CoA (monensin A). The meaA gene of this organism was cloned and sequenced and was shown to encode a putative 74-kDa protein with significant amino acid sequence identity to methylmalonyl-CoA mutase (MCM) (40%) and isobutyryl-CoA mutase (ICM) large subunit (36%) and small subunit (52%) from the same organism. The predicted C terminus of MeaA contains structural features highly conserved in all coenzyme B12-dependent mutases. Plasmid-based expression of meaA from the ermE* promoter in the S. cinnamonensis C730.1 strain resulted in a decreased ratio of monensin A to monensin B, from 1:1 to 1:3. Conversely, this ratio increased to 4:1 in a meaA mutant, S. cinnamonensis WM2 (generated from the C730.1 strain by insertional inactivation of meaA by using the erythromycin resistance gene). In both of these experiments, the overall monensin titers were not significantly affected. Monensin titers, however, did decrease over 90% in an S. cinnamonensis WD2 strain (an icm meaA mutant). Monensin titers in the WD2 strain were restored to at least wild-type levels by plasmid-based expression of the meaA gene or the Amycolatopsis mediterranei mutAB genes (encoding MCM). In contrast, growth of the WD2 strain in the presence of 0.8 M valine led only to a partial restoration (<25%) of monensin titers. These results demonstrate that the meaA gene product is significantly involved in methylmalonyl-CoA production in S. cinnamonensis and that under the tested conditions the presence of both MeaA and ICM is crucial for monensin production in the WD2 strain. These results also indicate that valine degradation, implicated in providing methylmalonyl-CoA precursors for many polyketide biosynthetic processes, does not do so to a significant degree for monensin biosynthesis in the WD2 mutant.

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Project description:Bimolecular rate constants have been measured for reactions that involve hydrogen atom transfer (HAT) from hydroxylamines to nitroxyl radicals, using the stable radicals TEMPO(*) (2,2,6,6-tetramethylpiperidine-1-oxyl radical), 4-oxo-TEMPO(*) (2,2,6,6-tetramethyl-4-oxo-piperidine-1-oxyl radical), di-tert-butylnitroxyl ((t)Bu(2)NO(*)), and the hydroxylamines TEMPO-H, 4-oxo-TEMPO-H, 4-MeO-TEMPO-H (2,2,6,6-tetramethyl-N-hydroxy-4-methoxy-piperidine), and (t)Bu(2)NOH. The reactions have been monitored by UV-vis stopped-flow methods, using the different optical spectra of the nitroxyl radicals. The HAT reactions all have |DeltaG (o)| < or = 1.4 kcal mol(-1) and therefore are close to self-exchange reactions. The reaction of 4-oxo-TEMPO(*) + TEMPO-H --> 4-oxo-TEMPO-H + TEMPO(*) occurs with k(2H,MeCN) = 10 +/- 1 M(-1) s(-1) in MeCN at 298 K (K(2H,MeCN) = 4.5 +/- 1.8). Surprisingly, the rate constant for the analogous deuterium atom transfer reaction is much slower: k(2D,MeCN) = 0.44 +/- 0.05 M(-1) s(-1) with k(2H,MeCN)/k(2D,MeCN) = 23 +/- 3 at 298 K. The same large kinetic isotope effect (KIE) is found in CH(2)Cl(2), 23 +/- 4, suggesting that the large KIE is not caused by solvent dynamics or hydrogen bonding to solvent. The related reaction of 4-oxo-TEMPO(*) with 4-MeO-TEMPO-H(D) also has a large KIE, k(3H)/k(3D) = 21 +/- 3 in MeCN. For these three reactions, the E(aD) - E(aH) values, between 0.3 +/- 0.6 and 1.3 +/- 0.6 kcal mol(-1), and the log(A(H)/A(D)) values, between 0.5 +/- 0.7 and 1.1 +/- 0.6, indicate that hydrogen tunneling plays an important role. The related reaction of (t)Bu(2)NO(*) + TEMPO-H(D) in MeCN has a large KIE, 16 +/- 3 in MeCN, and very unusual isotopic activation parameters, E(aD) - E(aH) = -2.6 +/- 0.4 and log(A(H)/A(D)) = 3.1 +/- 0.6. Computational studies, using POLYRATE, also indicate substantial tunneling in the (CH(3))(2)NO(*) + (CH(3))(2)NOH model reaction for the experimental self-exchange processes. Additional calculations on TEMPO((*)/H), (t)Bu(2)NO((*)/H), and Ph(2)NO((*)/H) self-exchange reactions reveal why the phenyl groups make the last of these reactions several orders of magnitude faster than the first two. By inference, the calculations also suggest why tunneling appears to be more important in the self-exchange reactions of dialkylhydroxylamines than of arylhydroxylamines.

Dataset Information

Coupling of hydrogenic tunneling to active-site motion in the hydrogen radical transfer catalyzed by a coenzyme B12-dependent mutase.

Publications

Coupling of hydrogenic tunneling to active-site motion in the hydrogen radical transfer catalyzed by a coenzyme B12-dependent mutase.

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