Molecular recognition in a post-translational modification of exceptional specificity. Mutants of the biotinylated domain of acetyl-CoA carboxylase defective in recognition by biotin protein ligase.
We have used localized mutagenesis of the biotin domain of the Escherichia coli biotin carboxyl carrier protein coupled with a genetic selection to identify regions of the domain having a role in interactions with the modifying enzyme, biotin protein ligase. We purified several singly substituted mutant biotin domains that showed reduced biotinylation in vivo and characterized these proteins in vitro. This approach has allowed us to distinguish putative biotin protein ligase interaction mutations from structurally defective proteins. Two mutant proteins with glutamate to lysine substitutions (at residues 119 or 147) behaved as authentic ligase interaction mutants. The E119K protein was virtually inactive as a substrate for biotin protein ligase, whereas the E147K protein could be biotinylated, albeit poorly. Neither substitution affected the overall structure of the domain, assayed by disulfide dimer formation and trypsin resistance. Substitutions of the highly conserved glycine residues at positions 133 and 143 or at a key hydrophobic core residue, Val-146, gave structurally unstable proteins.
Chapman-Smith
A
A
Department of Biochemistry, University of Adelaide, Adelaide, South Australia 5005, Australia.
Morris
T W
TW
Wallace
J C
JC
Cronan
J E
JE
D000103:Acetyl-CoA Carboxylase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D054889:Fatty Acid Synthase, Type II; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010957:Plasmids; D011485:Protein Binding; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; D054889:Fatty Acid Synthase, Type II; D019731:Carbon-Nitrogen Ligases; C027739:biotin-(acetyl-CoA carboxylase)synthetase; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein