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Hum Mutat. 2010 Sep;31(9):1033-42. doi: 10.1002/humu.21307.

Functional and structural analysis of five mutations identified in methylmalonic aciduria cblB type.

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Centro de Diagnéstico de Enfermedades Moleculares, Centro de Biología Molecular-SO UAM-CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, Madrid, Spain/Centro de Investigación Biomédica en Red de Enfermedades Raras, ISCIII, Madrid, Spain.


ATP:cob(I)alamin adenosyltransferase (ATR, E.C. converts reduced cob(I)alamin to the adenosylcobalamin cofactor. Mutations in the MMAB gene encoding ATR are responsible for the cblB type methylmalonic aciduria. Here we report the functional analysis of five cblB mutations to determine the underlying molecular basis of the dysfunction. The transcriptional profile along with minigenes analysis revealed that c.584G>A, c.349-1G>C, and c.290G>A affect the splicing process. Wild-type ATR and the p.I96T (c.287T>C) and p.R191W (c.571C>T) mutant proteins were expressed in a prokaryote and a eukaryotic expression systems. The p.I96T protein was enzymatically active with a K(M) for ATP and K(D) for cob(I)alamin similar to wild-type enzyme, but exhibited a 40% reduction in specific activity. Both p.I96T and p.R191W mutant proteins are less stable than the wild-type protein, with increased stability when expressed under permissive folding conditions. Analysis of the oligomeric state of both mutants showed a structural defect for p.I96T and also a significant impact on the amount of recovered mutant protein that was more pronounced for p.R191W that, along with the structural analysis, suggest they might be misfolded. These results could serve as a basis for the implementation of pharmacological therapies aimed at increasing the residual activity of this type of mutations.

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