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首页> 外文期刊>Biochemistry >Tertiary and quaternary structures of photoreactive Fe-type nitrile hydratase from Rhodococcus sp. N-771: roles of hydration water molecules in stabilizing the structures and the structural origin of the substrate specificity of the enzyme.
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Tertiary and quaternary structures of photoreactive Fe-type nitrile hydratase from Rhodococcus sp. N-771: roles of hydration water molecules in stabilizing the structures and the structural origin of the substrate specificity of the enzyme.

机译：Rhodococcus sp。的光反应性Fe型腈水合酶的三级和四级结构。 N-771：水合水分子在稳定酶底物特异性的结构和结构起源中的作用。

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The crystal structure analysis of the Fe-type nitrile hydratase from Rhodococcus sp. N-771 revealed the unique structure of the enzyme composed of the alpha- and beta-subunits and the unprecedented structure of the non-heme iron active center [Nagashima, S., et al. (1998) Nat. Struct. Biol. 5, 347-351]. A number of hydration water molecules were identified both in the interior and at the exterior of the enzyme. The study presented here investigated the roles of the hydration water molecules in stabilizing the tertiary and the quaternary structures of the enzyme, based on the crystal structure and the results from a laser light scattering experiment for the enzyme in solution. Seventy-six hydration water molecules between the two subunits significantly contribute to the alphabeta heterodimer formation by making up the surface shape, forming extensive networks of hydrogen bonds, and moderating the surface charge of the beta-subunit. In particular, 20 hydration water molecules form the extensive networks of hydrogen bonds stabilizing the unique structure of the active center. The amino acid residues hydrogen-bonded to those hydration water molecules are highly conserved among all known nitrile hydratases and even in the homologous enzyme, thiocyanate hydrolase, suggesting the structural conservation of the water molecules in the NHase family. The crystallographic asymmetric unit contained two heterodimers connected by 50 hydration water molecules. The heterotetramer formation in crystallization was clearly explained by the concentration-dependent aggregation state of NHase found in the light scattering measurement. The measurement proved that the dimer-tetramer equilibrium shifted toward the heterotetramer dominant state in the concentration range of 10(-2)-1.0 mg/mL. In the tetramer dominant state, 50 water molecules likely glue the two heterodimers together as observed in the crystal structure. Because NHase exhibits a high abundance in bacterial cells, the result suggests that the heterotetramer is physiologically relevant. In addition, it was revealed that the substrate specificity of this enzyme, recognizing small aliphatic substrates rather than aromatic ones, came from the narrowness of the entrance channel from the bulk solvent to the active center. This finding may give a clue for changing the substrate specificity of the enzyme. Under the crystallization condition described here, one 1,4-dioxane molecule plugged the channel. Through spectroscopic and crystallographic experiments, we found that the molecule prevented the dissociation of the endogenous NO molecule from the active center even when the crystal was exposed to light.
机译：红球菌Fe型腈水合酶的晶体结构分析。 N-771揭示了由α-和β-亚基组成的酶的独特结构，以及非血红素铁活性中心的空前结构[Nagashima，S.，等。 （1998）Nat。结构。生物学5，347-351]。在酶的内部和外部都发现了许多水合水分子。此处的研究基于晶体结构和溶液中酶的激光散射实验结果，研究了水合水分子在稳定酶的三级和四级结构中的作用。两个亚基之间的76个水合水分子通过补足表面形状，形成广泛的氢键网络并调节β亚基的表面电荷，显着促进了字母异二聚体的形成。特别是20个水合水分子形成了广泛的氢键网络，稳定了活性中心的独特结构。氢键合到那些水合水分子上的氨基酸残基在所有已知的腈水合酶中甚至在同源酶硫氰酸盐水解酶中都是高度保守的，这表明NHase家族中水分子的结构保守性。晶体学不对称单元包含通过50个水合水分子连接的两个异二聚体。结晶中的异四聚体形成可以通过光散射测量中发现的浓度依赖性的NHase聚集态清楚地解释。测量证明，在10（-2）-1.0 mg / mL的浓度范围内，二聚体-四聚体平衡向异四聚体显性态转移。在四聚体主导状态下，如晶体结构中所观察到的那样，有50个水分子可能将两个异二聚体粘合在一起。因为NHase在细菌细胞中表现出很高的丰度，所以结果表明异四聚体在生理上是相关的。另外，发现该酶的底物特异性是识别小的脂肪族底物而不是芳香族底物，其原因是从本体溶剂到活性中心的进入通道狭窄。该发现可以为改变酶的底物特异性提供线索。在此处描述的结晶条件下，一个1,4-二恶烷分子堵塞了通道。通过光谱和晶体学实验，我们发现即使在晶体暴露于光的情况下，该分子也能阻止内源性NO分子从活性中心解离。

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