Siezen R J, de Vos W M, Leunissen J A, Dijkstra B W
Department of Biophysical Chemistry, NIZO, Ede, The Netherlands.
Protein Eng. 1991 Oct;4(7):719-37. doi: 10.1093/protein/4.7.719.
Subtilases are members of the family of subtilisin-like serine proteases. Presently, greater than 50 subtilases are known, greater than 40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase and proteinase K). The mature enzymes contain up to 1775 residues, with N-terminal catalytic domains ranging from 268 to 511 residues, and signal and/or activation-peptides ranging from 27 to 280 residues. Several members contain C-terminal extensions, relative to the subtilisins, which display additional properties such as sequence repeats, processing sites and membrane anchor segments. Multiple sequence alignment of the N-terminal catalytic domains allows the definition of two main classes of subtilases. A structurally conserved framework of 191 core residues has been defined from a comparison of the four known three-dimensional structures. Eighteen of these core residues are highly conserved, nine of which are glycines. While the alpha-helix and beta-sheet secondary structure elements show considerable sequence homology, this is less so for peptide loops that connect the core secondary structure elements. These loops can vary in length by greater than 150 residues. While the core three-dimensional structure is conserved, insertions and deletions are preferentially confined to surface loops. From the known three-dimensional structures various predictions are made for the other subtilases concerning essential conserved residues, allowable amino acid substitutions, disulphide bonds, Ca(2+)-binding sites, substrate-binding site residues, ionic and aromatic interactions, proteolytically susceptible surface loops, etc. These predictions form a basis for protein engineering of members of the subtilase family, for which no three-dimensional structure is known.
枯草杆菌蛋白酶是枯草杆菌蛋白酶样丝氨酸蛋白酶家族的成员。目前，已知的枯草杆菌蛋白酶超过50种，其中40多种具有完整的氨基酸序列。我们比较了这些序列以及现有的三维结构（枯草杆菌蛋白酶BPN'、枯草杆菌蛋白酶卡尔伯格、嗜热菌蛋白酶和蛋白酶K）。成熟的酶含有多达1775个残基，N端催化结构域的长度在268至511个残基之间，信号肽和/或激活肽的长度在27至280个残基之间。相对于枯草杆菌蛋白酶，一些成员含有C端延伸，这些延伸具有额外的特性，如序列重复、加工位点和膜锚定片段。N端催化结构域的多序列比对可以定义两类主要的枯草杆菌蛋白酶。通过比较四个已知的三维结构，确定了一个由191个核心残基组成的结构保守框架。其中18个核心残基高度保守，9个是甘氨酸。虽然α-螺旋和β-折叠二级结构元件显示出相当程度的序列同源性，但连接核心二级结构元件的肽环的同源性较低。这些环的长度变化可以超过150个残基。虽然核心三维结构是保守的，但插入和缺失优先局限于表面环。根据已知的三维结构，对其他枯草杆菌蛋白酶在必需保守残基、允许的氨基酸取代、二硫键、Ca(2+)结合位点、底物结合位点残基、离子和芳香族相互作用、蛋白水解敏感表面环等方面进行了各种预测。这些预测为枯草杆菌蛋白酶家族中三维结构未知的成员的蛋白质工程提供了基础。
