Aparicio J F, Caffrey P, Marsden A F, Staunton J, Leadlay P F
Cambridge Centre for Molecular Recognition, University of Cambridge, United Kingdom.
J Biol Chem. 1994 Mar 18;269(11):8524-8.
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.
利用有限蛋白酶解和活性位点标记技术，对来自糖多孢红霉菌的红霉素聚酮合酶中6-脱氧红霉内酯B合酶1组分的结构域进行了研究。使用胰蛋白酶、弹性蛋白酶、谷氨酸蛋白酶C和精氨酸蛋白酶C切割该多酶，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳评估所得片段的大小。通过N端序列分析确定片段在一级结构中的位置。切割模式遵循先前基于序列比对预测的结构域边界，但许多预测的结构域间区域即使在最严苛的条件下也未被切割。初步蛋白酶解产生三个大片段：一个包含酰基转移酶-酰基载体蛋白双结构域的N端片段（约60 kDa）；一个包含酮合酶-酰基转移酶双结构域的中央片段（约90 kDa）；以及一个包含多酶其余六个结构域（包括第三个酰基转移酶）的C端片段（约220 kDa）。完整的多酶在凝胶过滤中表现为分子量660 kDa的二聚体；C端片段保持二聚体状态。然而，N端和中央片段似乎是单体形式。多酶蛋白酶解后，发现N端双结构域在与[14C]丙酰辅酶A孵育后被特异性标记，为其作为丙酸起始单元“装载结构域”的假定作用提供了首个证据。相比之下，另外两个片段被[14C]甲基丙二酰辅酶A特异性酰化，表明另外两个酰基转移酶在蛋白酶解后仍保持酶活性。
