Bekri M A, Desair J, Keijers V, Proost P, Searle-van Leeuwen M, Vanderleyden J, Vande Broek A
F. A. Janssens Laboratory of Genetics, Catholic University of Leuven, 3001 Heverlee, Belgium.
J Bacteriol. 1999 Apr;181(8):2440-7. doi: 10.1128/JB.181.8.2440-2447.1999.
The pelA gene from the N2-fixing plant-associated bacterium Azospirillum irakense, encoding a pectate lyase, was isolated by heterologous expression in Escherichia coli. Nucleotide sequence analysis of the region containing pelA indicated an open reading frame of 1,296 bp, coding for a preprotein of 432 amino acids with a typical amino-terminal signal peptide of 24 amino acids. N-terminal amino acid sequencing confirmed the processing of the protein in E. coli at the signal peptidase cleavage site predicted by nucleotide sequence analysis. Analysis of the amino acid sequence of PelA revealed no homology to other known pectinases, indicating that PelA belongs to a new pectate lyase family. PelA macerates potato tuber tissue, has an alkaline pH optimum, and requires Ca2+ for its activity. Of several divalent cations tested, none could substitute for Ca2+. Methyl-esterified pectin (with a degree of esterification up to 93%) and polygalacturonate can be used as substrates. Characterization of the degradation products formed upon incubation with polygalacturonate indicated that PelA is an endo-pectate lyase generating unsaturated digalacturonide as the major end product. Regulation of pelA expression was studied by means of a translational pelA-gusA fusion. Transcription of this fusion is low under all growth conditions tested and is dependent on the growth phase. In addition, pelA expression was found to be induced by pectin. An A. irakense pelA::Tn5 mutant still displayed pectate lyase activity, suggesting the presence of multiple pectate lyase genes in A. irakense.
通过在大肠杆菌中进行异源表达，从与固氮植物相关的细菌伊拉克固氮螺菌中分离出了编码果胶酸裂解酶的pelA基因。对包含pelA的区域进行核苷酸序列分析，结果表明存在一个1296 bp的开放阅读框，编码一个由432个氨基酸组成的前体蛋白，该前体蛋白带有一个由24个氨基酸组成的典型氨基末端信号肽。氨基末端氨基酸测序证实了该蛋白在大肠杆菌中是在核苷酸序列分析预测的信号肽酶切割位点进行加工的。对PelA氨基酸序列的分析表明，它与其他已知的果胶酶没有同源性，这表明PelA属于一个新的果胶酸裂解酶家族。PelA能使马铃薯块茎组织浸软，最适pH值呈碱性，且其活性需要Ca2+。在所测试的几种二价阳离子中，没有一种能够替代Ca2+。甲基酯化果胶（酯化度高达93%）和聚半乳糖醛酸可以用作底物。与聚半乳糖醛酸一起孵育后形成的降解产物的特性表明，PelA是一种内切果胶酸裂解酶，主要终产物是不饱和二半乳糖醛酸。通过翻译融合的pelA - gusA研究了pelA表达的调控。在所有测试的生长条件下，这种融合体的转录水平都很低，并且依赖于生长阶段。此外，发现果胶可诱导pelA的表达。伊拉克固氮螺菌的pelA::Tn5突变体仍表现出果胶酸裂解酶活性，这表明伊拉克固氮螺菌中存在多个果胶酸裂解酶基因。
