Black W J, Quinn F D, Tompkins L S
Department of Microbiology and Immunology, Stanford University, California 94305.
J Bacteriol. 1990 May;172(5):2608-13. doi: 10.1128/jb.172.5.2608-2613.1990.
The sequence of the structural gene encoding the Legionella pneumophila extracellular zinc metalloprotease has been determined and was found to possess a single large open reading frame (ORF) of 1,629 nucleotides (nt). This ORF was preceded by consensus promoter (TTAACT . . . 17 nt . . . TATAAC) and ribosome-binding (TAAGGAG) sequences. The deduced polypeptide contained a putative signal sequence and a total of 543 amino acid residues with a computed molecular size of 60,775 daltons, substantially larger than the observed 38,000 daltons of the native and recombinant proteins. A homology search revealed extensive amino acid identity with Pseudomonas aeruginosa elastase, a protein that is also encoded by an ORF substantially larger than that predicted for the mature size of the protein. The structural identity between the L. pneumophila protease and P. aeruginosa elastase was most pronounced in the regions forming the enzymatic active site of elastase. Amino acid residues constituting the active-site cleft of elastase were greater than 75% conserved. Elastase residues that interact with and mediate proteolysis of substrate peptides were 100% conserved. Competitive inhibitors of elastase and the structurally and functionally related thermolysin (phosphoramidon and a phosphoramidate analog, Z-GlyP(O)Leu-Ala), were shown to be equally potent at inhibiting the proteolytic activity of the L. pneumophila protease. These inhibitor studies along with the amino acid sequence similarities provide strong evidence that the L. pneumophila protease and P. aeruginosa elastase share a similar molecular mechanism of proteolysis.
编码嗜肺军团菌细胞外锌金属蛋白酶的结构基因序列已被确定，发现其具有一个1629个核苷酸（nt）的单一大型开放阅读框（ORF）。该ORF之前有共有启动子（TTAACT……17 nt……TATAAC）和核糖体结合（TAAGGAG）序列。推导的多肽包含一个推定的信号序列，共有543个氨基酸残基，计算的分子大小为60775道尔顿，大大大于天然和重组蛋白观察到的38000道尔顿。同源性搜索显示与铜绿假单胞菌弹性蛋白酶有广泛的氨基酸同一性，铜绿假单胞菌弹性蛋白酶也是由一个比预测的成熟蛋白大小的ORF大得多的ORF编码的。嗜肺军团菌蛋白酶和铜绿假单胞菌弹性蛋白酶之间的结构同一性在形成弹性蛋白酶酶活性位点的区域最为明显。构成弹性蛋白酶活性位点裂缝的氨基酸残基保守性大于75%。与底物肽相互作用并介导其蛋白水解的弹性蛋白酶残基100%保守。弹性蛋白酶和结构与功能相关的嗜热菌蛋白酶（膦酰脒酮和一种膦酸酯类似物，Z-GlyP（O）Leu-Ala）的竞争性抑制剂，被证明在抑制嗜肺军团菌蛋白酶的蛋白水解活性方面同样有效。这些抑制剂研究以及氨基酸序列相似性提供了强有力的证据，表明嗜肺军团菌蛋白酶和铜绿假单胞菌弹性蛋白酶具有相似的蛋白水解分子机制。
