Ega3 from the fungal pathogen Aspergillus fumigatus is an endo-α-1,4-galactosaminidase that disrupts microbial biofilms
Ega3 from the fungal pathogen Aspergillus fumigatus is an endo-α-1,4-galactosaminidase that disrupts microbial biofilms
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DOI：
10.1074/jbc.ra119.009910
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发表时间：
2019-09-13
影响因子：
4.8
通讯作者：
Howell, P. Lynne
中科院分区：
文献类型：
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作者：
Bamford, Natalie C.;Le Mauff, Francois;Howell, P. Lynne
烟曲霉是一种机会性真菌病原体，可引起慢性和急性侵袭性感染。半乳糖氨基半乳糖(GAG)是烟曲霉菌生物膜基质的重要组成部分，也是一个关键的毒力因子。GAG是一种由半乳糖和GalNAc组成的异构线性α-1，4-连接胞外多糖，分泌后部分脱乙酰化。一个由五个共表达的基因组成的簇与Gag的生物合成和修饰有关。该簇中的一个基因ega3被注释为编码一个可能的α-1，4-半乳糖胺酶，属于糖苷水解酶家族114(GH114)。在这里，我们证明了重组Ega3是一种活性的糖苷水解酶，可以在纳摩尔浓度下破坏依赖Gag的烟曲霉菌和依赖PEL多糖的铜绿假单胞菌生物膜。通过MS和功能分析，我们证明Ega3是一种内切作用的α-1，4-半乳糖胺酶，其活性依赖于保守的酸性残基Asp-189和Glu-247。对apo Ega3和Ega3-半乳糖胺络合物的X射线晶体结构分析，在1.76和2.09埃分辨率下，显示了一个修饰的(β/α)(8)折叠和一个深的电负性裂隙，当配体结合时，它被封顶形成隧道。我们的结构分析和电子对接研究也揭示了氨基半乳糖专一性和底物结合在-2到+1结合亚基上的分子决定因素。这项研究的发现增加了对GH114家族的结构和机制的理解，该家族有600个成员，由植物和机会人类病原体编码，以及工业上使用的细菌和真菌编码。
Aspergillus fumigatus is an opportunistic fungal pathogen that causes both chronic and acute invasive infections. Galactosaminogalactan (GAG) is an integral component of the A. fumigatus biofilm matrix and a key virulence factor. GAG is a heterogeneous linear alpha-1,4-linked exopolysaccharide of galactose and GalNAc that is partially deacetylated after secretion. A cluster of five co-expressed genes has been linked to GAG biosynthesis and modification. One gene in this cluster, ega3, is annotated as encoding a putative alpha-1,4-galactosaminidase belonging to glycoside hydrolase family 114 (GH114). Herein, we show that recombinant Ega3 is an active glycoside hydrolase that disrupts GAG-dependent A. fumigatus and Pel polysaccharide-dependent Pseudomonas aeruginosa biofilms at nanomolar concentrations. Using MS and functional assays, we demonstrate that Ega3 is an endo-acting alpha-1,4-galactosaminidase whose activity depends on the conserved acidic residues, Asp-189 and Glu-247. X-ray crystallographic structural analysis of the apo Ega3 and an Ega3-galactosamine complex, at 1.76 and 2.09 angstrom resolutions, revealed a modified (beta/alpha)(8)-fold with a deep electronegative cleft, which upon ligand binding is capped to form a tunnel. Our structural analysis coupled with in silico docking studies also uncovered the molecular determinants for galactosamine specificity and substrate binding at the -2 to +1 binding subsites. The findings in this study increase the structural and mechanistic understanding of the GH114 family, which has >600 members encoded by plant and opportunistic human pathogens, as well as in industrially used bacteria and fungi.