Active-site residues are critical for the folding and stability of methylamine dehydrogenase.
Active-site residues are critical for the folding and stability of methylamine dehydrogenase.
复制标题
活性位点残基对于甲胺脱氢酶的折叠和稳定性至关重要。
DOI：
10.1093/protein/14.9.675
复制
发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Davidson,VL
中科院分区：
文献类型：
--
作者：
Sun,D;Jones,LH;Mathews,FS;Davidson,VL
使用定点诱变来改变脱氮副球菌的甲胺脱氢酶（MADH）的活性位点残基。 MADH β 亚基的四个与色氨酸色氨酸醌 (TTQ) 辅基非常接近的残基被修饰。 MADH 的晶体结构揭示了这些残基中的每一个都参与与其他活性位点残基、TTQ 或水的氢键相互作用。相对保守的突变去除了 Thr122、Tyr119、Asp76 和 Asp32 侧链上的潜在活性氧，均导致 MADH 产生水平大大降低或检测不到。 MADH 水平的降低是通过使用 MADH β 亚基特异性抗血清对粗提物进行活性测定和蛋白质印迹来确定的。在表达 D76N、T122A 和 T122C MADH 突变体的细胞提取物中未检测到活性或交叉反应蛋白。表达 D32N、Y119F、Y119E 和 Y119K MADH 突变体的细胞产生极低水平的活性 MADH。从细胞提取物中纯化 Y119F 和 D32N 突变体，发现其稳定性明显低于野生型 MADH。仅 T122S MADH 突变体以接近野生型的水平产生。讨论了这些氨基酸残基在稳定 MADH β 亚基的异常结构特征、蛋白质折叠和 TTQ 生物合成中的可能作用。
Site-directed mutagenesis was used to alter active-site residues of methylamine dehydrogenase (MADH) fromParacoccus denitrificans. Four residues of the β subunit of MADH which are in close proximity to the tryptophan tryptophylquinone (TTQ) prosthetic group were modified. The crystal structure of MADH reveals that each of these residues participates in hydrogen bonding interactions with other active-site residues, TTQ or water. Relatively conservative mutations which removed the potentially reactive oxygens on the side chains of Thr122, Tyr119, Asp76 and Asp32 each resulted in greatly reduced or undetectable levels of MADH production. The reduction of MADH levels was determined by assays of activity and Western blots of crude extracts with antisera specific for the MADH β subunit. No activity or cross-reactive protein was detected in extracts of cells expressing D76N, T122A and T122C MADH mutants. Very low levels of active MADH were produced by cells expressing D32N, Y119F, Y119E and Y119K MADH mutants. The Y119F and D32N mutants were purified from cell extracts and found to be significantly less stable than wild-type MADH. Only the T122S MADH mutant was produced at near wild-type levels. Possible roles for these amino acid residues in stabilizing unusual structural features of the MADH β subunit, protein folding and TTQ biosynthesis are discussed.