Overexpression and characterization of a prolyl endopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus
Overexpression and characterization of a prolyl endopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus
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超嗜热古菌激烈火球菌脯氨酰内肽酶的过度表达和表征
DOI：
10.1128/jb.179.11.3613-3618.1997
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发表时间：
1997
影响因子：
3.2
通讯作者：
H. Schreier
中科院分区：
文献类型：
--
作者：
Valerie J. Harwood;Jackie D. Denson;K. A. Robinson;H. Schreier
从嗜热古细菌furiococcus中克隆了一种麦芽糖调控的mlr-2基因，该基因与细菌和真核生物的脯氨酸内肽酶（PEPase）同源。重组细胞的提取物能够水解酶底物ZGPpNA (benzyloxycarbonyl-Gly-Pro-p-nitroanilide, ZGPpNA)，最适温度在85 ~ 90℃之间。纯化的PEPase变性凝胶电泳显示酶活性与一个70 kda的蛋白相关，这与mlr-2序列的预测一致。然而，凝胶渗透研究获得了59 kDa的表观分子质量。除了ZGPpNA （K（Mapp）为53微米）外，PEPase能够水解偶氮酪蛋白，尽管速率较低。当ZGPpNA被羧基肽酶A和B、凝乳胰蛋白酶、枯草杆菌蛋白酶和中性内肽酶的底物取代时，未检测到活性。N-[N-（l- 3-反式carboxirane -2-羰基）- l- leu]-agmatine （E-64）和toyl - l- lys氯甲基酮对PEPase活性无抑制作用。苯甲基磺酰氟和二蛋白A均抑制ZGPpNA的切割，后者具有竞争性（K（lapp）为343微米）。在100℃时，酶对十二烷基硫酸钠处理表现出一定的耐受性。随着时间的推移，PEPase的稳定性取决于蛋白质浓度；在高于65℃的温度下，稀释后的样品在24 h后仍保持大部分活性，而浓缩后的样品活性明显下降。这种减少被发现部分是由于自身蛋白水解。从金黄色葡萄球菌中部分纯化的PEPase与在大肠杆菌中过表达的酶具有相同的最适温度、分子量和动力学特性。在麦芽糖存在下培养的真菌提取物的PEPase活性比不含麦芽糖的培养物高约7倍。从麦芽糖培养物中获得的澄清培养基中无法检测到活性。我们得出结论，mlr-2，现在称为prpA，编码PEPase；这种蛋白酶的生理作用目前尚不清楚。
The maltose-regulated mlr-2 gene from the hyperthermophilic archaeon Pyrococcus furiosus having homology to bacterial and eukaryal prolyl endopeptidase (PEPase) was cloned and overexpressed in Escherichia coli. Extracts from recombinant cells were capable of hydrolyzing the PEPase substrate benzyloxycarbonyl-Gly-Pro-p-nitroanilide (ZGPpNA) with a temperature optimum between 85 and 90 degrees C. Denaturing gel electrophoresis of purified PEPase showed that enzyme activity was associated with a 70-kDa protein, which is consistent with that predicted from the mlr-2 sequence. However, an apparent molecular mass of 59 kDa was obtained from gel permeation studies. In addition to ZGPpNA (K(Mapp) of 53 microM), PEPase was capable of hydrolyzing azocasein, although at a low rate. No activity was detected when ZGPpNA was replaced by substrates for carboxypeptidase A and B, chymotrypsin, subtilisin, and neutral endopeptidase. N-[N-(L-3-trans-Carboxirane-2-carbonyl)-L-Leu]-agmatine (E-64) and tosyl-L-Lys chloromethyl ketone did not inhibit PEPase activity. Both phenylmethylsulfonyl fluoride and diprotin A inhibited ZGPpNA cleavage, the latter doing so competitively (K(lapp) of 343 microM). At 100 degrees C, the enzyme displayed some tolerance to sodium dodecyl sulfate treatment. Stability of PEPase over time was dependent on protein concentration; at temperatures above 65 degrees C, dilute samples retained most of their activity after 24 h while the activity of concentrated preparations diminished significantly. This decrease was found to be due, in part, to autoproteolysis. Partially purified PEPase from P. furiosus exhibited the same temperature optimum, molecular weight, and kinetic characteristics as the enzyme overexpressed in E. coli. Extracts from P. furiosus cultures grown in the presence of maltose were approximately sevenfold greater in PEPase activity than those grown without maltose. Activity could not be detected in clarified medium obtained from maltose-grown cultures. We conclude that mlr-2, now called prpA, encodes PEPase; the physiological role of this protease is presently unknown.
DOI：
10.3109/10409239309082572
发表时间：
1993
影响因子：
6.5
作者：
A. Yaron;F. Naider
通讯作者：
A. Yaron;F. Naider