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

✍ Scribed by Ana Jorge-Finnigan; Cristina Aguado; Rocio Sánchez-Alcudia; David Abia; Eva Richard; Begoña Merinero; Alejandra Gámez; Ruma Banerjee; Lourdes R. Desviat; Magdalena Ugarte; Belen Pérez

Publisher
John Wiley and Sons
Year
2010
Tongue
English
Weight
477 KB
Volume
31
Category
Article
ISSN
1059-7794

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✦ Synopsis

ATP:cob(I)alamin adenosyltransferase (ATR, E.C.2.5.1.17) 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.584G4A, c.349-1G4C, and c.290G4A affect the splicing process. Wild-type ATR and the p.I96T (c.287T4C) and p.R191W (c.571C4T) 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 wildtype 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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