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Functional and structural analysis of five mutations identified in methylmalonic aciduria cbIB type
Author(s) -
JorgeFinnigan Ana,
Aguado Cristina,
SánchezAlcudia Rocio,
Abia David,
Richard Eva,
Merinero Begoña,
Gámez Alejandra,
Banerjee Ruma,
Desviat Lourdes R.,
Ugarte Magdalena,
Pérez Belen
Publication year - 2010
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.21307
Subject(s) - biology , mutant , wild type , mutant protein , biochemistry , microbiology and biotechnology , mutation , functional analysis , rna splicing , gene , genetics , rna
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.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. Hum Mutat 31:1033–1042, 2010. © 2010 Wiley‐Liss, Inc.