Characterization and structural modeling of a novel thermostable glycine oxidase from Geabacilus kausuiphilus HTA426
Characterization and structural modeling of a novel thermostable glycine oxidase from Geabacilus kausuiphilus HTA426
复制标题
DOI：
10.1002/prot.21690
复制
发表时间：
2008-03-01
影响因子：
2.9
通讯作者：
Sanchez-Ferrer, Alvaro
中科院分区：
文献类型：
--
作者：
Martinez-Martinez, Irene;Navarro-Fernandez, Jose;Sanchez-Ferrer, Alvaro
来自嗜热土芽孢杆菌HTA 426（GOXK）的甘氨酸氧化酶是含有非共价结合的FAD的43 kDa单体黄素酶。该酶的诱导导致了一种完全可溶的蛋白质的表达，该蛋白质具有比先前报道的来自B的GOX更高的比活性。枯草芽孢杆菌（GOXB）。对这种新型GOXK的动力学性质的研究揭示了所分析的大多数底物的最低Km值，但D-脯氨酸除外，其保持类似的值并且具有所报道的最高V-max值。V-max/K-M比保持GOXK对小尺寸胺（如甘氨酸、肌氨酸、N-乙基甘氨酸和甘氨酸乙酯）的底物偏好。GOXK在60-70 ℃和碱性介质（pH 6-9.5）中表现出良好的稳定性。通过序列比对和比较GOXK和GOXB之间的变化，建立了推测的三维结构模型，发现了可能与底物特异性有关的残基以及对催化活性至关重要的残基，并将GOXK和GOXB的可能拓扑结构进行了比较，发现了构建四聚体寡聚体的单体之间的相互作用发生了变化。
Glycine oxidase from Geobacillus kaustophilus HTA426 (GOXK) is a 43 kDa monomer flavoenzyme containing noncovalently bound FAD. The induction of the enzyme resulted in the expression of a fully soluble protein with higher specific activity than those previously reported for GOX from B. subtilis (GOXB). A study of the kinetic properties of this novel GOXK revealed the lowest Km values for most of the substrates analyzed, with the exception Of D-proline which kept a similar value and had the highest V-max value reported. The V-max/K-M ratio maintained a substrate preference of GOXK for amines of small size, like glycine, sarcosine, N-ethyl-glycine, and glycine-ethylester. GOXK presented good stability at 60-70 degrees C and in alkaline media (pH 6-9.5). The putative tridimensional structure was modeled by sequence alignment and by comparing the changes between GOXK and GOXB, and the residues that could be responsible for the substrate specificity as well as those essential for the catalytic activity were found. The comparison between the possible topology of GOXK with that of GOXB showed changes at the putative interactions between monomers for the building of the tetrameric oligomerization.