Citation:

CCS Chemistry:金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的?

[J]. CCS Chemistry, ;2020, 2(0): 946-954. doi: 10.31635/ccschem.020.202000219 shu

CCS Chemistry:金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的?

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  •    中山大学夏炜、香港大学孙红哲课题组通过解析金黄色葡萄球菌醛基脱氢酶纯蛋白以及复合物晶体结构,发现其特有的“C-helix”结构域在结合特异性底物过程中发生的构象变化,提出了金黄色葡萄球菌醛基脱氢酶识别特异性底物的分子门控机制,并通过一系列生化实验辅助验证。



       

       金黄色葡萄球菌是一种常见人类病原菌,可引起一系列感染性疾病,由于抗生素的滥用,产生了许多耐药性的金黄色葡萄球菌,而相对于开发新的抗生素,发展新的抗感染疗法对于对抗病毒感染更为有效。超过90%的金黄色葡萄球菌临床分离株会产生一种金黄色的含有30个碳(C30)的长链类胡萝卜素分子,称为葡萄球菌黄素,该色素可作为抗氧化剂提高细菌对于活性氧的耐受能力。因此,阻断其生物合成途径是一项重要的工作。


       葡萄球菌黄素的生物合成路径需要一系列催化酶的参与(图1)。其中,醛基脱氢酶(SaAldH)是最近被发现的参与色素合成的酶类,其功能是催化长链不饱和醛4,4’- diaponeurosporen-4-al生成对应的羧酸。近期,中山大学夏炜课题组以及香港大学孙红哲课题组报道了SaAldH纯蛋白以及其与特异性底物复合物的晶体结构,在分子层面揭示了SaAldH识别特异性底物——多不饱和长碳链脂肪醛的门控机制。


    图1 葡萄球菌黄素合成通路示意图


       从纯蛋白结构中,作者发现SaAldH二聚体除了保守的催化结构域、NAD结合结构域以及桥连结构域以外,在C端还具有独特的“C-helix”结构,类似于人源脂肪醛脱氢酶(FALDH)中的“gatekeeper helix”。将纯蛋白和复合物晶体结构对比后,发现“C-helix”在结合特异性底物之后发生了明显的构象变化(图2),朝着底物通道入口的方向转动了10.6°的夹角,从而使得氨基酸残基F456和F457的侧链插入到底物通道入口处,破坏了纯蛋白状态下F456、F457以及Y92形成的T型π-π堆叠相互作用。此外“C-helix”之前的loop区域的氨基酸V441和H442也向着底物通道入口的方向靠近,从而实现了底物口袋的闭合。因此作者们提出“C-helix”充当了重要的门控作用,在纯蛋白状态下,酶活口袋处于“开合”状态,允许底物的进入,而当酶结合了特异性底物之后,“C-helix”发生构象变化,在通道入口处形成位阻,锁住底物,使得酶活口袋处于“闭合”状态。


    图2 SaAldH的“open-closed”构象变化

       通过对SaAldH和特异性底物之间的相互作用进行分析,作者们发现特异性底物与SaAldH底物结合通道中的氨基酸残基形成广泛的疏水相互作用。另外,酶序列上Y116与底物的2-甲基形成π–σ相互作用,F457与底物末端的两个甲基形成疏水相互作用,暗示着“C-helix”同时也参与了对特异性底物的识别(图3)。



    图3 SaAldH与其特异性底物相互作用分析

       最后,作者们通过体外酶活实验、过氧化氢耐受实验以及巨噬细胞吞噬实验对关键氨基酸位点进行验证,证实结构中看到的关键性位点确实参与了特异性底物的识别(图4)。



    图4 关键氨基酸参与特异性底物识别的生化实验验证

       此项研究得到了国家自然科学基金、香港研究资助局、中国教育部以及中央高校基础研究经费的资助。该工作以research article的形式发表在CCS Chemistry,并在官网“Just Published”栏目上线。


    文章详情:

    Structural Insight into the Substrate Gating Mechanism by Staphylococcus aureus Aldehyde Dehydrogenase

    Xuan Tao , Zhemin Zhang , Xiao Zhang , Hongyan Li, Hongzhe Sun *, Zong-Wan Mao& Wei Xia *

    Citation:CCS Chem. 2020, 2, 946–954

    Link:https://doi.org/10.31635/ccschem.020.202000219


    图2 SaAldH的“open-closed”构象变化
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