Herrler M, Bang H, Marahiel M A
Philipps-Universität Marburg, Fachbereich Chemie/Biochemie, Germany.
Mol Microbiol. 1994 Mar;11(6):1073-83. doi: 10.1111/j.1365-2958.1994.tb00384.x.
Sequencing of N-terminal and internal peptide fragments of the purified 17 kDa Bacillus subtilis peptidyl-prolyl cis-trans isomerase (PPIase) revealed sequence identity to conserved regions of a number of eukaryotic and prokaryotic cyclophilins. Using two oligonucleotide primers corresponding to the N-terminus and a highly conserved internal amino acid sequence, polymerase chain reactions (PCR) with B. subtilis genomic DNA were carried out. The resultant PCR fragment of 335 bp was cloned, sequenced and subsequently used as a probe for screening a lambda Zap II gene library of B. subtilis. Two overlapping positive clones of 5 and 7 kb containing the B. subtilis PPIase gene (ppiB), which is 432 bp in length and encodes a protein of 144 amino acid residues, were identified and two distinct transcriptional initiation sites at the 5' end of ppiB were mapped. The entire region (35 kb) between spoVA and serA was recently sequenced in B. subtilis, and an open reading frame (ORF) that encodes a putative peptidyl-prolyl cis-trans isomerase at about 210 degrees on the B. subtilis genetic map was located. This putative PPIase is identical to PPiB. We have overexpressed the ppiB gene in Escherichia coli, purified the encoded protein to apparent homology and shown that it exhibits PPIase activity. In addition, the recombinant PPiB shows a significant inhibition of PPIase activity by cyclosporin A (CsA) at a level comparable to that observed for the B. subtilis enzyme. Interestingly the B. subtilis PPIase shows about 40% identity to eukaryotic PPIases and less similarity to those of Gram-negative bacteria (27-32% identity). Like other interruption mutants of yeast and Neurospora, which lack a functional cyclophilin gene, a B. subtilis mutant containing ppiB::cat, a cat-interrupted copy of ppiB in the chromosome, is viable.
对纯化的17 kDa枯草芽孢杆菌肽基脯氨酰顺反异构酶（PPIase）的N端和内部肽片段进行测序，结果显示其与许多真核和原核亲环蛋白的保守区域具有序列同一性。使用对应于N端和高度保守的内部氨基酸序列的两个寡核苷酸引物，以枯草芽孢杆菌基因组DNA进行聚合酶链反应（PCR）。将得到的335 bp PCR片段进行克隆、测序，随后用作筛选枯草芽孢杆菌λZap II基因文库的探针。鉴定出两个重叠的阳性克隆，大小分别为5 kb和7 kb，包含长度为432 bp、编码144个氨基酸残基蛋白质的枯草芽孢杆菌PPIase基因（ppiB），并确定了ppiB 5'端的两个不同转录起始位点。最近对枯草芽孢杆菌中spoVA和serA之间的整个区域（35 kb）进行了测序，并在枯草芽孢杆菌遗传图谱上约210度处定位了一个编码推定肽基脯氨酰顺反异构酶的开放阅读框（ORF）。这个推定的PPIase与PPiB相同。我们已在大肠杆菌中过表达ppiB基因，将编码的蛋白质纯化至表观同源性，并表明它具有PPIase活性。此外，重组PPiB显示环孢菌素A（CsA）对PPIase活性有显著抑制，其水平与枯草芽孢杆菌酶相当。有趣的是，枯草芽孢杆菌PPIase与真核PPIase的同一性约为40%，与革兰氏阴性菌的相似性较低（同一性为27 - 32%）。与缺乏功能性亲环蛋白基因的酵母和粗糙脉孢菌的其他中断突变体一样，枯草芽孢杆菌中含有ppiB::cat（染色体中ppiB的cat中断拷贝）的突变体是有活力的。
