Conformational change in the active site of Streptococcal unsaturated glucuronyl hydrolase through site-directed mutagenesis at Asp-115
Conformational change in the active site of Streptococcal unsaturated glucuronyl hydrolase through site-directed mutagenesis at Asp-115
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通过 Asp-115 定点突变改变链球菌不饱和葡萄糖醛酸水解酶活性位点的构象
DOI：
10.1007/s10930-016-9673-y
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
and Wataru Hashimoto
中科院分区：
文献类型：
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作者：
Yusuke Nakamichi;Sayoko Oiki;Bunzo Mikami;Kousaku Murata;and Wataru Hashimoto
细菌不饱和葡萄糖醛酸水解酶（UGL）通过多糖裂解酶降解由哺乳动物细胞外基质糖胺聚糖产生的不饱和双糖。无乳链球菌（streptococcus agalactiaeUGL, SagUGL）的两个Asp残基Asp-115和Asp-175在其他细菌的ugl中是完全保守的，其中一个（SagUGL的Asp-175）作为一般的酸碱催化剂。另一个Asp （SagUGL的Asp-115）也会影响酶的活性，尽管其在酶反应中的作用尚未得到很好的理解。在这里，我们发现用Asn取代SagUGL中的Asp-115导致活性位点的构象变化。通过x射线晶体学分别在2.00和1.79 Å分辨率下测定了酶活性可忽略的SagUGL突变体D115N和D115N/K370S的三级结构。在这两个突变体中，Asn-115的侧链由于与包括Asp-175在内的几个残基相互作用形成氢键而发生了剧烈的移位。Asn-115和Asp-175之间的相互作用可能会阻止突变体以Asp-175作为酸性催化剂触发酶反应。
Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 ofStreptococcus agalactiaeUGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.