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 componen t of the erythromycin-producing polyketide synthase from Saccharopolys pora erythraea has been investigated using limited proteolysis and act ive-site labeling. Trypsin, elastase, endoproteinase Glu-C, and endopr oteinase Arg-C were used to cleave the multienzyme, and the sizes of t he resulting fragments were assessed by sodium dodecyl sulfate-polyacr ylamide gel electrophoresis. The location of fragments within the prim ary structure was established by N-terminal sequence analysis. The cle avage pattern followed domain boundaries previously predicted on the b asis of sequence alignments, but many predicted interdomain regions we re not cleaved, even under the harshest conditions used. Initial prote olysis generated three large fragments: an N-terminal fragment (about 60 kDa) housing an acyltransferase-acyl carrier protein di-domain; a c entral fragment (about 90 kDa) containing a ketosynthase-acyltransfera se di-domain; and a C-terminal fragment (about 220 kDa) containing the remaining six domains of the multienzyme, including the third acyltra nsferase. 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-doma in was found to be specifically labeled after incubation with [C-14]pr opionyl-CoA, providing the first evidence for its proposed role as a ' 'loading domain'' for the propionate starter unit. In contrast, the ot her two fragments were specifically acylated by [C-14]methylmalonyl-Co A, indicating that both the other two acyltransferases remain enzymati cally active after proteolysis.