Völkl P, Markiewicz P, Stetter K O, Miller J H
Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90024.
Protein Sci. 1994 Aug;3(8):1329-40. doi: 10.1002/pro.5560030819.
The hyperthermophilic archaeum Pyrobaculum aerophilum grows optimally at 100 degrees C and pH 7.0. Cell homogenates exhibit strong proteolytic activity within a temperature range of 80-130 degrees C. During an analysis of cDNA and genomic sequence tags, a genomic clone was recovered showing strong sequence homology to alkaline subtilisins of Bacillus sp. The total DNA sequence of the gene encoding the protease (named "aerolysin") was determined. Multiple sequence alignment with 15 different serine-type proteases showed greatest homology with subtilisins from gram-positive bacteria rather than archaeal or eukaryal serine proteases. Models of secondary and tertiary structure based on sequence alignments and the tertiary structures of subtilisin Carlsberg, BPN', thermitase, and protease K were generated for P. aerophilum subtilisin. This allowed identification of sites potentially contributing to the thermostability of the protein. One common transition put alanines at the beginning and end of surface alpha-helices. Aspartic acids were found at the N-terminus of several surface helices, possibly increasing stability by interacting with the helix dipole. Several of the substitutions in regions expected to form surface loops were adjacent to each other in the tertiary structure model.
嗜热古菌嗜气栖热袍菌在100℃和pH 7.0的条件下生长最佳。细胞匀浆在80 - 130℃的温度范围内表现出很强的蛋白水解活性。在对cDNA和基因组序列标签进行分析的过程中，获得了一个基因组克隆，该克隆与芽孢杆菌属的碱性枯草杆菌蛋白酶具有很强的序列同源性。确定了编码该蛋白酶（命名为“气溶素”）的基因的完整DNA序列。与15种不同的丝氨酸型蛋白酶进行多序列比对后发现，它与革兰氏阳性菌的枯草杆菌蛋白酶的同源性最高，而非古菌或真核生物的丝氨酸蛋白酶。基于序列比对以及枯草杆菌蛋白酶卡尔伯格、BPN'、嗜热菌蛋白酶和蛋白酶K的三级结构，构建了嗜气栖热袍菌枯草杆菌蛋白酶的二级和三级结构模型。这使得能够识别可能有助于该蛋白质热稳定性的位点。一个常见的转变是在表面α螺旋的起始和末端出现丙氨酸。在几个表面螺旋的N端发现了天冬氨酸，可能通过与螺旋偶极相互作用来增加稳定性。在三级结构模型中，预期形成表面环的区域中的几个取代彼此相邻。
