title: The roles of threonine-136 and glutamate-137 of human medium chain acyl-CoA dehydrogenase in FAD binding and peptide folding using site-directed mutagenesis: creation of an FAD-dependent mutant, T136D.
abstract: We studied the roles of Thr-136 (T136) and Glu-137 (E137) in the biogenesis of medium chain acyl-CoA dehydrogenase (MCAD) by altering the former to Ser (T136S), Asp (T136D), or Leu (T136L) and the latter to Asp (E137D), Gln (E137Q), or Lys (E137K). After import into mitochondria, T136S and E137D were assembled into the native tetramer as efficiently as the wild-type. The tetrameric assembly of four other variants with a nonconservative substitution was severely impaired. When expressed in Escherichia coli as the mature subunit, the amounts of the catalytically active forms of T136S and E137D were comparable to wild-type, whereas four nonconservative variants were lost as aggregates. Of these nonconservative variants, only T136D formed catalytically active tetramer when the culture broth and buffers were supplemented with riboflavin and FAD, respectively. Culturing T136L or E137K at a lower temperature (28 degreesC) did not increase the yield at all, suggesting the severity of disruption of biogenesis. These results, together with the previous crystallographic findings, indicate that the T136 hydroxyl is a major FAD-binding site, and that E137 carboxyl plays a key role in the beta-domain folding, through salt bridge formation with K164. These findings also support the notion that the isoalloxazine ring plays a critical role in the MCAD folding, presumably exerting nucleating effects.
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, 06510, USA. saijot@clin.med.tokushima-u.ac.jp
authors.forename: T
authors.initials: T
authors.lastname: Saijo
authors.forename: J J
authors.initials: JJ
authors.lastname: Kim
authors.forename: Y
authors.initials: Y
authors.lastname: Kuroda
authors.forename: K
authors.initials: K
authors.lastname: Tanaka
mesh_terms: D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000534:Alum Compounds; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D002850:Chromatography, Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005590:Fractional Precipitation; D015964:Gene Expression Regulation, Bacterial; D018698:Glutamic Acid / Q000235:genetics*; D006801:Humans; D008930:Mitochondria, Liver / Q000201:enzymology / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D051381:Rats; D012256:Riboflavin / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D013696:Temperature; D013912:Threonine / Q000235:genetics*
chemical_list: D000534:Alum Compounds; D005182:Flavin-Adenine Dinucleotide; D013912:Threonine; D001224:Aspartic Acid; C041524:aluminum sulfate; D018698:Glutamic Acid; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase; D012256:Riboflavin