Alternative pathways for radical dissipation in an active site mutant of B12-dependent methylmalonyl-CoA mutase

Biochemistry
Dominique Padovani, Ruma Banerjee

Abstract

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 x 10(4))-fold compared to wild-type enzyme, the K(M)app for the cofactor approximately 4-fold, and the cob(II)alamin concentration under steady-state turnover conditions approximately 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 analogues 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 c...Continue Reading

References

Dec 20, 1978·Biochimica Et Biophysica Acta·H RuppR Cammack
Jan 28, 1999·The Journal of Biological Chemistry·T Toraya, K Mori
May 2, 2003·Journal of the American Chemical Society·Monica D Vlasie, Ruma Banerjee
Sep 30, 2004·Journal of the American Chemical Society·Gregory M SandalaLeo Radom

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Citations

Dec 4, 2009·Proceedings of the National Academy of Sciences of the United States of America·Dominique Padovani, Ruma Banerjee
Jul 23, 2013·Nature Chemical Biology·Michael LofgrenRuma Banerjee
Jun 5, 2012·Journal of Zhejiang University. Science. B·Tóshiko Takahashi-IñiguezMaría Elena Flores
Apr 28, 2006·The Journal of Biological Chemistry·Dominique PadovaniRuma Banerjee
Sep 3, 2013·The Journal of Biological Chemistry·Michael LofgrenRuma Banerjee
Oct 24, 2017·Cell·Hongying ShenVamsi K Mootha

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