Tyrosine 89 Accelerates Co−Carbon Bond Homolysis in Methylmalonyl-CoA Mutase

Vlasie, Monica D.; Banerjee, Ruma

doi:10.1021/ja029420+

Cited by 41 publications

(63 citation statements)

References 20 publications

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“…Binding of substrate seems to trigger the barrel closure (Fig. 5B), which, in turn, causes Tyr-89 to swing toward the top of the AdoCbl corrin ring, presumably facilitating the homolytic cleavage of the Ado moiety to give Ado • (37,40). Thus, whereas MCM and 5,6-LAM have very similar structures, the way in which conformational changes are linked to substrate binding is different and takes advantage of the unique properties of the substrates.…”

Section: Discussionmentioning

confidence: 99%

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

Berkovitch

Behshad

Tang

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

105

109

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Lysine 5,6-aminomutase is an adenosylcobalamin and pyridoxal-5 -phosphate-dependent enzyme that catalyzes a 1,2 rearrangement of the terminal amino group of DL-lysine and of L-␤-lysine. We have solved the x-ray structure of a substrate-free form of lysine-5,6-aminomutase from Clostridium sticklandii. In this structure, a Rossmann domain covalently binds pyridoxal-5 -phosphate by means of lysine 144 and positions it into the putative active site of a neighboring triosephosphate isomerase barrel domain, while simultaneously positioning the other cofactor, adenosylcobalamin, Ϸ25 Å from the active site. In this mode of pyridoxal-5 -phosphate binding, the cofactor acts as an anchor, tethering the separate polypeptide chain of the Rossmann domain to the triosephosphate isomerase barrel domain. Upon substrate binding and transaldimination of the lysine-144 linkage, the Rossmann domain would be free to rotate and bring adenosylcobalamin, pyridoxal-5 -phosphate, and substrate into proximity. Thus, the structure embodies a locking mechanism to keep the adenosylcobalamin out of the active site and prevent radical generation in the absence of substrate.A denosylcobalamin (AdoCbl; coenzyme B 12 ) is nature's biochemical radical reservoir, capable of catalyzing challenging chemical reactions by way of H atom abstraction and the generation of free-radical intermediates (1-3). AdoCbldependent isomerases catalyze 1,2 shifts between an H atom and a functional group such as -OH, -NH 3 ϩ , -(CO)S-coenzyme A, or other carbon-based groups. The catalytic power of AdoCbl lies in the homolytic cleavage of its weak (Ϸ30 kcal͞mol) organometallic C-Co bond, formed between an octahedral Co(III) center with five N coordinations and a 5Ј-deoxyadenosyl (Ado) axial ligand. C-Co bond homolysis results in the transient formation of cob(II)alamin and 5Ј-deoxyadenosyl radical (Ado • ). Ado • abstracts an H atom from the substrate, forming a substrate radical and 5Ј-deoxyadenosine (AdoH). To close the catalytic cycle, substrate reabstracts the H atom from AdoH, and recombination of cob(II)alamin and Ado • accompanies product formation. Amazingly, the enzymatic rate of C-Co bond homolysis is enhanced by a factor of Ϸ10 12 over nonenzymatic homolysis (4, 5). AdoCbl-dependent isomerases are often present in catabolic pathways and can serve to rearrange the substrate's carbon skeleton and͞or functional groups for further degradation. One such pathway that operates in several bacterial species is the fermentation of lysine to yield acetate. Interestingly, the lysine fermentation pathway contains two analogous enzymes: lysine 5,6-aminomutase (5,6-LAM), which is AdoCbl-dependent (6, 7), and lysine 2,3-aminomutase (2,3-LAM), which is an S-adenosylmethionine (AdoMet or SAM)-dependent ironsulfur enzyme (8-10). Both enzymes require pyridoxal 5Ј-phosphate (PLP) (8, 11) in addition to AdoCbl or AdoMet, and both catalyze a 1,2 amino group shift with concomitant H atom migration (Fig. 1A). In 5,6-LAM, AdoCbl is the source of the transient Ado • , whereas, in 2,3-L...

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Section: Discussionmentioning

confidence: 99%

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

Berkovitch

Behshad

Tang

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

105

109

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“…The kinetic parameters for inactivation by allylmalonyl-CoA were obtained by UV-visible stopped-flow spectrophotometry by monitoring the formation of cob(II)alamin and H 2 OCbl. Holo-Y243A (9 μM after mixing) was rapidly mixed with various concentrations of allylmalonyl-CoA (7,14,21,30, 50 and 100 μM after mixing) in 50 mM potassium phosphate buffer, pH 7.5 at 20°C. Cob(II)alamin formation was monitored by a decrease in absorbance at 525 nm over a 0.5 sec period and H 2 OCbl formation was monitored by an increase at 351 nm over an 8 sec period.…”

Section: Uv-visible Absorption Spectroscopy Of Enzyme-substrate (Analmentioning

confidence: 99%

“…The hypothesis that Y89 functions as a molecular wedge that labilizes the Co-carbon bond, was derived from crystallographic data, which revealed that substrate-driven barrel closure results in a large motion of this residue and steric crowding above the corrin ring (9,11). The role of Y89 in labilizing the Co-carbon bond was supported by mutagenesis and kinetic studies, which also revealed its contribution to the rearrangement step (3,7).…”

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Alternative Pathways for Radical Dissipation in an Active Site Mutant of B₁₂-Dependent Methylmalonyl-CoA Mutase

Padovani

Banerjee

2006

Biochemistry

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Methylmalonyl-CoA mutase catalyzes the adenosylcobalamin-dependent rearrangement of (2R)-methylmalonyl-CoA to succinyl-CoA. The crystal structure of the enzyme reveals that Y243 is in van der Waals contact with the methyl group of the substrate and suggests a possible role for it in the stereochemical control of the reaction. This hypothesis was tested by designing a molecular hole by replacing the phenolic side chain of Y243 with the methyl group of alanine. The Y243A mutation lowered the catalytic efficiency >4 × 10 4 -fold compared to wild type enzyme, the K Mapp for the cofactor ~4-fold, and the cob(II)alamin concentration under steady-state turnover conditions ~2-fold. However, the mutation did not appear to lead to loss of the stereochemical preference for the substrate. The Y243A mutation is expected to create a cavity and should in principle, allow accommodation of bulkier substrates. To test this, we used ethylmalonyl-CoA and allylmalonyl-CoA, as alternate substrates. Surprisingly, both analogs resulted in suicidal inactivation albeit in an O 2 -dependent and O 2 -independent fashion, respectively. The inactivation by allylmalonyl-CoA was further investigated and revealed formation of cob(II)alamin at a ~1.5-fold higher rate than with wild-type mutase under single turnover conditions. Product analysis revealed a stoichiometric mixture of 5′-deoxyadenosine, aquocobalamin and allylmalonyl-CoA. Taken together, these results are consistent with an internal electron transfer from cob(II)alamin to the substrate analog radical. These studies serve to emphasize the fine control exerted by Y243 in the vicinity of the substrate to minimize radical extinction in side reactions.Methylmalonyl-CoA mutase catalyzes the reversible isomerization of methylmalonyl-CoA to succinyl-CoA and is dependent on 5′-deoxyadenosylcobalamin (AdoCbl) 1 or coenzyme B 12 for activity (Figure 1) (1). In this reaction, the AdoCbl cofactor is used as a radical reservoir that generates via homolysis of theCo-carbon bond, a pair of radicals: cob(II)alamin and a reactive 5′-deoxyadenosyl radical (Ado•). The latter abstracts a hydrogen atom from the substrate generating a substrate-centered radical that, in turn, rearranges to a product-centered radical. The remainder of the catalytic cycle is completed by reversal of the sequence of steps in the first half of the reaction (Figure 1).Two questions of long-standing interest regarding AdoCbl-dependent enzymes are how the inherent kinetic inertness of the Co-carbon bond is overcome to effect a trillion-fold acceleration in the homolysis rate (2) and how the high reactivity of radical intermediates are controlled to minimize side reactions.

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“…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.…”

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

Kitanishi

Cracan

Banerjee

2015

Journal of Biological Chemistry

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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 B 12 -dependent PCM was identified, cloned, and expressed and exhibited PCM activity. Significance: The newly discovered PCM could be useful in bioremediation and biosynthetic reactions.

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Tyrosine 89 Accelerates Co−Carbon Bond Homolysis in Methylmalonyl-CoA Mutase

Cited by 41 publications

References 20 publications

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

Alternative Pathways for Radical Dissipation in an Active Site Mutant of B₁₂-Dependent Methylmalonyl-CoA Mutase

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

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Tyrosine 89 Accelerates Co−Carbon Bond Homolysis in Methylmalonyl-CoA Mutase

Cited by 41 publications

References 20 publications

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

Alternative Pathways for Radical Dissipation in an Active Site Mutant of B12-Dependent Methylmalonyl-CoA Mutase

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

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Alternative Pathways for Radical Dissipation in an Active Site Mutant of B₁₂-Dependent Methylmalonyl-CoA Mutase