Ilari Andrea, Bonamore Alessandra, Franceschini Stefano, Fiorillo Annarita, Boffi Alberto, Colotti Gianni
Institute of Molecular Biology and Pathology, C.N.R., Rome, Italy.
Proteins. 2008 Jun;71(4):2065-75. doi: 10.1002/prot.21898.
The X-ray structure of monomeric N-methyltryptophan oxidase from Escherichia coli (MTOX) has been solved at 3.2 A resolution by molecular replacement methods using Bacillus sp. sarcosine oxidase structure (MSOX, 43% sequence identity) as search model. The analysis of the substrate binding site highlights the structural determinants that favour the accommodation of the bulky N-methyltryptophan residue in MTOX. In fact, although the nature and geometry of the catalytic residues within the first contact shell of the FAD moiety appear to be virtually superposable in MTOX and MSOX, the presence of a Thr residue in position 239 in MTOX (Met245 in MSOX) located at the entrance of the active site appears to play a key role for the recognition of the amino acid substrate side chain. Accordingly, a 15 fold increase in k(cat) and 100 fold decrease in K(m) for sarcosine as substrate has been achieved in MTOX upon T239M mutation, with a concomitant three-fold decrease in activity towards N-methyltryptophan. These data provide clear evidence for the presence of a catalytic core, common to the members of the methylaminoacid oxidase subfamily, and of a side chain recognition pocket, located at the entrance of the active site, that can be adjusted to host diverse aminoacids in the different enzyme species. The site involved in the covalent attachment of flavin has also been addressed by screening degenerate mutants in the relevant positions around Cys308-FAD linkage. Lys341 appears to be the key residue involved in flavin incorporation and covalent linkage.
利用芽孢杆菌属肌氨酸氧化酶结构（MSOX，序列同一性为43%）作为搜索模型，通过分子置换法以3.2埃的分辨率解析了来自大肠杆菌的单体N-甲基色氨酸氧化酶（MTOX）的X射线结构。对底物结合位点的分析突出了有利于在MTOX中容纳庞大的N-甲基色氨酸残基的结构决定因素。事实上，尽管在MTOX和MSOX中，黄素腺嘌呤二核苷酸（FAD）部分第一接触壳内催化残基的性质和几何结构几乎是可叠加的，但位于活性位点入口处的MTOX中239位的苏氨酸残基（MSOX中的Met245）的存在似乎对氨基酸底物侧链的识别起着关键作用。因此，在MTOX中，T239M突变使肌氨酸作为底物时的催化常数（k(cat)）增加了15倍，米氏常数（K(m)）降低了100倍，同时对N-甲基色氨酸的活性降低了三倍。这些数据清楚地证明了甲基氨基酸氧化酶亚家族成员共有的催化核心的存在，以及位于活性位点入口处的侧链识别口袋的存在，该口袋可以进行调整以容纳不同酶种中的各种氨基酸。通过筛选围绕半胱氨酸308-FAD连接的相关位置的简并突变体，也确定了参与黄素共价连接的位点。赖氨酸341似乎是参与黄素掺入和共价连接的关键残基。
