Nishiya Y, Imanaka T
Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., Fukui Prefecture, Japan.
Appl Environ Microbiol. 1996 Jul;62(7):2405-10. doi: 10.1128/aem.62.7.2405-2410.1996.
Sarcosine oxidase from Arthrobacter sp. TE1826 (SoxA) tightly binds with the coenzyme flavin adenine dinucleotide (FAD). The amino-terminal region of this enzyme was recognized as a part of the FAD-binding domain by homology search analysis. Comparison with other structurally well-known flavoproteins suggested that the aspartate residue at position 35 (D-35) and the motif sequence (six residues at positions 12 to 17) were important for the interaction with FAD. Site-directed mutagenesis of each position was performed, and mutant SoxAs were purified and characterized. When D-35 was substituted with glutamate, asparagine, and alanine, it was indicated that the carboxyl group of the side chain interacted with FAD. Changes in the enzyme-bound FAD were also observed from the altered spectral profiles. Thirteen mutant SoxAs were obtained by replacing amino acids in the motif sequence. Most of them showed inhibited or remarkably decreased sarcosine oxidase activity, and their spectral profiles were altered. However, some of them were reactivated by chloride ion. Their spectral profiles also became close to that of wild type in the presence of chloride ion. These results strongly suggest that the inhibition of interaction of enzyme with FAD was caused by the substitution in the motif and that it could be recovered under different conditions.
来自节杆菌属TE1826菌株的肌氨酸氧化酶（SoxA）与辅酶黄素腺嘌呤二核苷酸（FAD）紧密结合。通过同源搜索分析，该酶的氨基末端区域被认为是FAD结合结构域的一部分。与其他结构已知的黄素蛋白进行比较表明，第35位的天冬氨酸残基（D-35）和基序序列（第12至17位的六个残基）对于与FAD的相互作用很重要。对每个位置进行定点诱变，然后纯化并表征突变型SoxA。当D-35被谷氨酸、天冬酰胺和丙氨酸取代时，表明侧链的羧基与FAD相互作用。从改变的光谱图中也观察到酶结合FAD的变化。通过替换基序序列中的氨基酸获得了13种突变型SoxA。其中大多数表现出肌氨酸氧化酶活性受到抑制或显著降低，并且它们的光谱图发生了改变。然而，其中一些在氯离子存在下被重新激活。在氯离子存在下，它们的光谱图也变得接近野生型。这些结果有力地表明，基序中的取代导致了酶与FAD相互作用的抑制，并且在不同条件下可以恢复这种抑制作用。
