Structural basis for the core-mannan biosynthesis of cell wall fungal-type galactomannan in Aspergillus fumigatus
Structural basis for the core-mannan biosynthesis of cell wall fungal-type galactomannan in Aspergillus fumigatus
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DOI：
10.1074/jbc.ra120.013742
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发表时间：
2020-11-06
影响因子：
4.8
通讯作者：
Oka, Takuji
中科院分区：
文献类型：
--
作者：
Hira, Daisuke;Onoue, Takuya;Oka, Takuji
真菌细胞壁及其生物合成酶是新型抗真菌药物的潜在靶点。最近发现两种甘露糖转移酶，即核心-甘露聚糖合成酶A(CMSA/Ktr4)和核心-甘露聚糖合成酶B(CMSB/Ktr7)，在真菌半乳甘露聚糖的核心-甘露聚糖生物合成中发挥作用。CMSA/Ktr4是一种α-(1-gt；2)-甘露糖基转移酶，负责真菌型半乳甘露聚糖的生物合成，覆盖在烟曲霉的细胞表面。在侵袭性曲霉病的小鼠模型中，CMSA/Ktr4基因突变的菌株表现出强烈的菌丝伸长和分生孢子抑制，同时毒力降低，表明CMSA/Ktr4是一个潜在的新的抗真菌候选基因。在本研究中，我们用X射线结晶学方法测定了CMSA/Ktr4的可溶性催化域的三维结构，分辨率为1.95埃，以及酶和Mn2+/GDP络合物的分辨率为1.90埃。CMSA/Ktr4蛋白不仅含有与供体底物GDP-甘露糖高度保守的结合口袋，而且具有由N-末端和C-末端形成的独特的宽裂隙结构，有望识别受体底物-甘露聚糖链。基于这些晶体结构，我们还提出了以α-Man-(1->；6)-α-Man-(1-gt；2)-α-Man-ome为模型结构的对接和分子动力学模拟生成的酶-底物复合体的三维结构模型。这一预测的酶-底物复杂结构也得到了在Delta CMSA/ktr4 A.fumigatus细胞中表达的单一氨基酸替换CMSA/Ktr4突变体的研究结果的支持。综上所述，这些结果为开发用作药物和/或杀虫剂的特定α-甘露聚糖生物合成抑制剂提供了基本信息。
Fungal cell walls and their biosynthetic enzymes are potential targets for novel antifungal agents. Recently, two mannosyltransferases, namely core-mannan synthases A (CmsA/Ktr4) and B (CmsB/Ktr7), were found to play roles in the core-mannan biosynthesis of fungal-type galactomannan. CmsA/Ktr4 is an alpha-(1 -> 2)-mannosyltransferase responsible for alpha-(1 -> 2)-mannan biosynthesis in fungal-type galactomannan, which covers the cell surface of Aspergillus fumigatus. Strains with disrupted cmsA/ktr4 have been shown to exhibit strongly suppressed hyphal elongation and conidiation alongside reduced virulence in a mouse model of invasive aspergillosis, indicating that CmsA/Ktr4 is a potential novel antifungal candidate. In this study we present the 3D structures of the soluble catalytic domain of CmsA/Ktr4, as determined by X-ray crystallography at a resolution of 1.95 angstrom, as well as the enzyme and Mn2+/GDP complex to 1.90 angstrom resolution. The CmsA/Ktr4 protein not only contains a highly conserved binding pocket for the donor substrate, GDP-mannose, but also has a unique broad cleft structure formed by its N- and C-terminal regions and is expected to recognize the acceptor substrate, a mannan chain. Based on these crystal structures, we also present a 3D structural model of the enzyme-substrate complex generated using docking and molecular dynamics simulations with alpha-Man-(1 -> 6)-alpha-Man-(1 -> 2)-alpha-Man-OMe as the model structure for the acceptor substrate. This predicted enzyme-substrate complex structure is also supported by findings from single amino acid substitution CmsA/Ktr4 mutants expressed in Delta cmsA/ktr4 A. fumigatus cells. Taken together, these results provide basic information for developing specific alpha-mannan biosynthesis inhibitors for use as pharmaceuticals and/or pesticides.