Murooka Y, Ikeda R
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Japan.
J Biol Chem. 1989 Oct 15;264(29):17524-31.
We constructed, by site-directed mutagenesis, a mutant pullulanase gene in which the cysteine residue in a pentapeptide sequence, Leu16-Leu-Ser-Gly-Cys20 within the NH2-terminal region of pullulanase from Klebsiella aerogenes, is replaced by serine (Ser20). The modification, processing, and subcellular localization of the mutant pullulanase were studied. Labeling studies with [3H]palmitate and immunoprecipitation with mouse antiserum raised against pullulanase showed that the wild form of both the extracellular and intracellular pullulanases contained lipids, whereas the mutant enzyme was not modified with lipids. Only the Cys20 was modified with glyceryl lipids. The bulk of the mutant pullulanase was located in the periplasm, but a portion of the unmodified, mutant pullulanase was secreted into the medium. Mutant pullulanases from the extracellular and the periplasm were purified and their NH2-terminal sequences were determined. Both the mutant pullulanases were cleaved between residues of Ser13 and Leu14 which is 6-amino acid residues upstream of the lipid modified pullulanase cleavage site. This new cleavage was resistant to globomycin, an inhibitor of the prolipoprotein signal peptidase of Escherichia coli. These results indicate that the pentapeptide sequence plays an important role in maturation and translocation of pullulanase in K. aerogenes. However, the modification of pullulanase with lipids seems to be not essential for export of the enzyme across the outer membrane.
我们通过定点诱变构建了一个突变支链淀粉酶基因，其中产气克雷伯菌支链淀粉酶NH2末端区域五肽序列Leu16 - Leu - Ser - Gly - Cys20中的半胱氨酸残基被丝氨酸（Ser20）取代。对突变支链淀粉酶的修饰、加工及亚细胞定位进行了研究。用[3H]棕榈酸进行标记研究以及用针对支链淀粉酶产生的小鼠抗血清进行免疫沉淀表明，细胞外和细胞内支链淀粉酶的野生型均含有脂质，而突变酶未被脂质修饰。只有Cys20被甘油脂质修饰。大部分突变支链淀粉酶位于周质中，但有一部分未修饰的突变支链淀粉酶分泌到了培养基中。对细胞外和周质中的突变支链淀粉酶进行了纯化并测定了其NH2末端序列。两种突变支链淀粉酶均在Ser13和Leu14残基之间被切割，该切割位点比脂质修饰的支链淀粉酶切割位点上游多6个氨基酸残基。这种新的切割对球霉素具有抗性，球霉素是大肠杆菌前脂蛋白信号肽酶的抑制剂。这些结果表明，该五肽序列在产气克雷伯菌支链淀粉酶的成熟和转运中起重要作用。然而，支链淀粉酶的脂质修饰似乎对于该酶跨外膜输出并非必不可少。
