Limited proteolysis and active-site studies of the first multienzyme component of the erythromycin-producing polyketide synthase.
The domain structure of the 6-deoxyerythronolide B synthase 1 component of the erythromycin-producing polyketide synthase from Saccharopolyspora erythraea has been investigated using limited proteolysis and active-site labeling. Trypsin, elastase, endoproteinase Glu-C, and endoproteinase Arg-C were used to cleave the multienzyme, and the sizes of the resulting fragments were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The location of fragments within the primary structure was established by N-terminal sequence analysis. The cleavage pattern followed domain boundaries previously predicted on the basis of sequence alignments, but many predicted interdomain regions were not cleaved, even under the harshest conditions used. Initial proteolysis generated three large fragments: an N-terminal fragment (about 60 kDa) housing an acyltransferase-acyl carrier protein di-domain; a central fragment (about 90 kDa) containing a ketosynthase-acyltransferase di-domain; and a C-terminal fragment (about 220 kDa) containing the remaining six domains of the multienzyme, including the third acyltransferase. The intact multienzyme behaves as a dimer of molecular mass 660 kDa on gel filtration; and the C-terminal fragment remains dimeric. However, the N-terminal and central fragments appear to be monomeric species. After proteolysis of the multienzyme, the N-terminal di-domain was found to be specifically labeled after incubation with [14C]propionyl-CoA, providing the first evidence for its proposed role as a "loading domain" for the propionate starter unit. In contrast, the other two fragments were specifically acylated by [14C]methylmalonyl-CoA, indicating that both the other two acyltransferases remain enzymatically active after proteolysis.
Aparicio
J F
JF
Cambridge Centre for Molecular Recognition, University of Cambridge, United Kingdom.
Caffrey
P
P
Marsden
A F
AF
Staunton
J
J
Leadlay
P F
PF
D000595:Amino Acid Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D004917:Erythromycin / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016930:Saccharopolyspora / Q000201:enzymology*
D009097:Multienzyme Complexes; D010446:Peptide Fragments; D004917:Erythromycin; D010450:Endopeptidases