Expression of recombinant human methylmalonyl-CoA mutase: in primary mut fibroblasts and Saccharomyces cerevisiae.
Methylmalonyl-CoA mutase is an adenosylcobalamin-dependent enzyme which catalyzes isomerization of methylmalonyl-CoA to succinyl-CoA. Previous reports have described cloning and sequencing of a cDNA for human methylmalonyl-CoA mutase. This clone does not express an active apoenzyme after gene transfer into primary MCM-deficient fibroblasts and contains several sequences which differ from the consensus sequence of other cDNA clones. We describe reconstruction of a functional MCM cDNA and expression of recombinant enzyme activity in primary fibroblasts and Saccharomyces cerevisiae. This consensus human MCM cDNA is capable of complementing the inherited defect in mut MMA and overexpressing an enzyme in yeast with kinetic properties indistinguishable from the enzyme in murine or human tissues.
Andrews
E
E
Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030.
Jansen
R
R
Crane
A M
AM
Cholin
S
S
McDonnell
D
D
Ledley
F D
FD
D000214:Acyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003038:Cobamides / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis; D005347:Fibroblasts / Q000201:enzymology; D018014:Gene Transfer Techniques; D006801:Humans; D007700:Kinetics; D008765:Methylmalonyl-CoA Mutase / Q000096:biosynthesis* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000214:Acyl Coenzyme A; D003038:Cobamides; D018076:DNA, Complementary; D011994:Recombinant Proteins; C015357:methylmalonyl-coenzyme A; D008765:Methylmalonyl-CoA Mutase; C000913:cobamamide