a Campbell Cancer Research Institute, Ontario Cancer Institute, Princess Margaret Hospital, University Health Network , Toronto , Ontario , M5G 2C4 , Canada.
J Biomol Struct Dyn. 2013 Oct;31(10):1057-65. doi: 10.1080/07391102.2012.719111. Epub 2012 Oct 2.
The nitrilases include a variety of enzymes with functional specificities of nitrilase, amidase, and hydrolase reactions. The crystal structure of the uncharacterized protein SA0302 from the pathogenic microorganism Staphylococcus aureus is solved at 1.7 Å resolution. The protein contains 261 amino acids and presents a four-layer αββα sandwich with a chain topology similar to that of a few known CN-hydrolase folds. In the crystal, the proteins are arranged as dimers whose monomers are related by a pseudo twofold rotation symmetry axis. Analysis of the sequences and structures of CN-hydrolases with known 3D structures shows that SA0302 definitely is a member of Branch 10 (Nit and NitFhit) of the nitrilase superfamily. Enzyme activities and substrate specificities of members of this branch are not yet characterized, in contrast to those of the members of Branches 1-9. Although the sequence identities between Branch 10 members are rather low, less than 30%, five conserved regions are common in this subfamily. Three of them contain functionally important catalytic residues, and the two other newly characterized ones are associated with crucial intramolecular and intermolecular interactions. Sequence homology of the area near the active site shows clearly that the catalytic triad of SA0302 is Glu41-Lys110-Cys146. We suggest also that the active site includes a fourth residue, the closely located Glu119. Despite an extensive similarity with other Nit-family structural folds, SA0302 displays an important difference. Protein loop 111-122, which follows the catalytic Lys110, is reduced to half the number of amino acids found in other Nit-family members. This leaves the active site fully accessible to solvent and substrates. We have identified conservative sequence motifs around the three core catalytic residues, which are inherent solely to Branch 10 of the nitrilase superfamily. On the basis of these new sequence fingerprints, 10 previously uncharacterized proteins also could be assigned to this hydrolase subfamily. An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:19.
腈水解酶包括具有腈酶、酰胺酶和水解酶反应功能特异性的各种酶。致病性微生物金黄色葡萄球菌中未鉴定蛋白 SA0302 的晶体结构在 1.7Å分辨率下得到解决。该蛋白包含 261 个氨基酸，呈现出四层αββα三明治结构，其链拓扑结构类似于几个已知的 CN-水解酶折叠。在晶体中，蛋白质排列为二聚体，其单体通过拟二倍旋转对称轴相关。对具有已知 3D 结构的 CN-水解酶序列和结构的分析表明，SA0302 绝对是腈水解酶超家族分支 10（Nit 和 NitFhit）的成员。与分支 1-9 的成员不同，该分支成员的酶活性和底物特异性尚未得到表征。尽管分支 10 成员之间的序列同一性相当低，不到 30%，但在这个亚家族中共有五个保守区域。其中三个包含功能上重要的催化残基，另外两个新鉴定的残基与关键的分子内和分子间相互作用相关。活性位点附近区域的序列同源性清楚地表明，SA0302 的催化三联体为 Glu41-Lys110-Cys146。我们还建议，活性位点还包括第四个残基，即紧密相邻的 Glu119。尽管与其他 Nit 家族结构折叠具有广泛的相似性，但 SA0302 显示出一个重要的区别。紧随催化 Lys110 的蛋白质环 111-122 减少到其他 Nit 家族成员数量的一半。这使得活性位点完全可以与溶剂和底物接触。我们已经确定了在三个核心催化残基周围的保守序列基序，这些基序仅存在于腈水解酶超家族的分支 10 中。基于这些新的序列指纹，还可以将 10 个以前未鉴定的蛋白质分配到这个水解酶亚家族中。动画交互式 3D 补充（I3DC）可在 Proteopedia 上获得，网址为 http://proteopedia.org/w/Journal:JBSD:19。
