CLUSTERING OF MUTATIONS IN METHYLMALONYL COA MUTASE ASSOCIATED WITH MUT(-) METHYLMALONIC ACIDEMIA

Mutations have been described in human methylmalonyl CoA mutase (MCM) that exhibit partial defects in enzyme activity, including cobalamin-d ependent (i.e., mut(-)) or interallelic complementation. This work des cribes mutations in cells from four patients, three of whom exhibit a cobalamin-dependent phenotype and all four of whom exhibit interalleli c complementation. Four novel mutations (R694W, G648D, G630E, and G626 C) are identified that cluster near the carboxyl terminus of the prote in, a region close to another mut(-) mutation (G717V). Each of these m utations was shown to express a phenotype congruent with that of the p arental cell line, after transfection into mut(0) fibroblasts, and eac h exhibits interallelic complementation in cotransfection assays with clones bearing a R93H mutation. The activity of mutant enzymes express ed in Saccharomyces cerevisiae parallels the residual activity of the parental cell lines and exhibits novel sensitivities to pH and salt. T he clustering of these mutations identifies a region of MCM that most likely represents the cobalamin-binding domain. The location of this d omain, as well as the pattern of sequence preservation between the hom ologous human and Probionobacterium shermanii enzymes, suggests a mech anism for interallelic complementation in which the cobalamin-binding defect is complemented in trans from the heterologous subunits of the dimer.