Characterization of wild-type and an active site mutant of human medium chain acyl-CoA dehydrogenase after expression in Escherichia coli.
The cDNA of human medium chain acyl-CoA dehydrogenase (MCADH) was modified by in vitro mutagenesis, and the sequence encoding the mature form of MCADH was introduced into an inducible expression plasmid. We observed synthesis of the protein in Escherichia coli cells transformed with this plasmid with measurable MCADH enzyme activity in cell extracts. Glutamic acid 376, which has been proposed by Powell and Thorpe (Powell, P. J., and Thorpe, J. (1988) Biochemistry 27, 8022-8028) as an essential residue and the proton-abstracting base at the active site of the enzyme, was mutated to glutamine. After expression in bacteria of this plasmid, the corresponding extracts show no detectable MCADH activity, although mutant MCADH-protein production was detected by protein immunoblots. The mature enzyme and the Gln376 mutant were purified to apparent homogeneity. The wild-type enzyme is a yellow protein due to the content of stoichiometric FAD and had a specific activity which is 50% of MCADH purified from pig kidney. The Gln376 mutant is devoid of activity (less than 0.02% that of wild type, expressed enzyme) and is green because of bound CoA persulfide. Properties of the mutant enzyme suggest that the Glu376----Gln change specifically affects substrate binding. These results prove that Glu376 plays an important role in the initial step of dehydrogenation catalysis.
Bross
P
P
Faculty of Biology, University of Konstanz, Federal Republic of Germany.
Engst
S
S
Strauss
A W
AW
Kelly
D P
DP
Rasched
I
I
Ghisla
S
S
D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005971:Glutamates; D018698:Glutamic Acid; D005973:Glutamine; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D013053:Spectrophotometry; D013552:Swine
D005971:Glutamates; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D005973:Glutamine; D018698:Glutamic Acid; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase