Expression and Kinetic Characterization of Methylmalonyl-CoA Mutase from Patients with the Mut− Phenotype: Evidence for Naturally Occurring Interallelic Complementation

• Published in: Human molecular genetics Vol. 6; no. 9; pp. 1457 - 1464
• Main Authors: Janata, Jirί, Kogekar, Nandini, Fenton, Wayne A.
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01-09-1997
• Subjects: Biological and medical sciences; Cell Line; Errors of metabolism; Escherichia coli - enzymology; Escherichia coli - genetics; Gene Expression; Genetic Complementation Test; Genetic Vectors; Humans; Medical sciences; Metabolic diseases; Methylmalonyl-CoA Mutase - analysis; Methylmalonyl-CoA Mutase - genetics; Methylmalonyl-CoA Mutase - metabolism; Miscellaneous hereditary metabolic disorders; Molecular Sequence Data; Phenotype; Point Mutation; Polymerase Chain Reaction; Human; Enzyme; Deficiency; Metabolic diseases; Gene expression; Enzymopathy; Genetic disease; Acidemia; Mitochondria; Isomerases; Enzymatic activity; Gene; Methylmalonic acid; Intramolecular transferases; Genetics; Complementation; Kinetics; Mutation; Methylmalonyl-CoA mutase
• ISSN: 0964-6906; 1460-2083; 1460-2083
• DOI: 10.1093/hmg/6.9.1457

l-Methylmalonyl-CoA mutase (MUT) is an adenosylcobalamin (AdoCbl)-requiring mitochondrial matrix enzyme that catalyzes the isomerization of l-methylmalonyl-CoA to succinyl-CoA. Inherited defects in the gene encoding this enzyme result in the mut forms of methylmalonic acidemia. Expression of mature human MUT cDNA in Escherichia coli at a post-induction cultivation temperature of 12°C, rather than 37°C, led to the folding of the majority of the synthesized protein to a soluble form, with an activity of 0.2–0.3 U/mg protein in the cell-free extract, 10–15 times higher than that in human liver homogenate. Six missense mutations, producing the amino acid changes G94V, Y231N, R369H, G623R, H678R and G717V, were detected in MUT cDNA of patients suffering from the mut− form of methylmalonic acidemia, resulting from defective AdoCbl binding. Two (G623R and G717V) had been reported in other patients. Three (G94V, Y231N and R369H) are the first changes in the NH2-terminal part of the enzyme reported to cause the mut− phenotype. Enzymes with the mutations were individually expressed, and their kinetic parameters were generally in accord with published biochemical data from extracts of fibroblasts from these patients. The mutations increased the Km for AdoCbl by 40- to 900-fold, while Vmax values varied from 0.2% to nearly 100% of that of wild-type protein. In one case of a doubly heterozygous cell line, however, neither of the constituent mutant enzymes had a Km corresponding to the lower of the two estimated from the extract data. This finding may reflect the natural occurrence of interallelic complementation in vivo in this cell line.