Biotin protein ligase from Saccharomyces cerevisiae. The N-terminal domain is required for complete activity.
Catalytically active biotin protein ligase from Saccharomyces cerevisiae (EC 6.3.4.15) was overexpressed in Escherichia coli and purified to near homogeneity in three steps. Kinetic analysis demonstrated that the substrates ATP, biotin, and the biotin-accepting protein bind in an ordered manner in the reaction mechanism. Treatment with any of three proteases of differing specificity in vitro revealed that the sequence between residues 240 and 260 was extremely sensitive to proteolysis, suggesting that it forms an exposed linker between an N-terminal 27-kDa domain and the C-terminal 50-kDa domain containing the active site. The protease susceptibility of this linker region was considerably reduced in the presence of ATP and biotin. A second protease-sensitive sequence, located in the presumptive catalytic site, was protected against digestion by the substrates. Expression of N-terminally truncated variants of the yeast enzyme failed to complement E. coli strains defective in biotin protein ligase activity. In vitro assays performed with purified N-terminally truncated enzyme revealed that removal of the N-terminal domain reduced BPL activity by greater than 3500-fold. Our data indicate that both the N-terminal domain and the C-terminal domain containing the active site are necessary for complete catalytic function.
Polyak
S W
SW
Department of Biochemistry, University of Adelaide, Adelaide, South Australia SA 5005, Australia.
Chapman-Smith
A
A
Brautigan
P J
PJ
Wallace
J C
JC
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry* / Q000235:genetics; D011756:Diphosphates / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D005816:Genetic Complementation Test; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017384:Sequence Deletion; D014157:Transcription Factors
D001426:Bacterial Proteins; D011756:Diphosphates; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D010450:Endopeptidases; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli