BACKGROUND The pathogenic yeast Candida albicans can proliferate in environments with different carbon dioxide concentrations thanks to the carbonic anhydrase CaNce103p, which accelerates spontaneous conversion of carbon dioxide to bicarbonate and vice versa. Without functional CaNce103p, C. albicans cannot survive in atmospheric air. CaNce103p falls into the β-carbonic anhydrase class, along with its ortholog ScNce103p from Saccharomyces cerevisiae. The crystal structure of CaNce103p is of interest because this enzyme is a potential target for surface disinfectants. RESULTS Recombinant CaNce103p was prepared in E. coli, and its crystal structure was determined at 2.2 Å resolution. CaNce103p forms a homotetramer organized as a dimer of dimers, in which the dimerization and tetramerization surfaces are perpendicular. Although the physiological role of CaNce103p is similar to that of ScNce103p from baker's yeast, on the structural level it more closely resembles carbonic anhydrase from the saprophytic fungus Sordaria macrospora, which is also tetrameric. Dimerization is mediated by two helices in the N-terminal domain of the subunits. The N-terminus of CaNce103p is flexible, and crystals were obtained only upon truncation of the first 29 amino acids. Analysis of CaNce103p variants truncated by 29, 48 and 61 amino acids showed that residues 30-48 are essential for dimerization. Each subunit contains a zinc atom in the active site and displays features characteristic of type I β-carbonic anhydrases. Zinc is tetrahedrally coordinated by one histidine residue, two cysteine residues and a molecule of β-mercaptoethanol originating from the crystallization buffer. The active sites are accessible via substrate tunnels, which are slightly longer and narrower than those observed in other fungal carbonic anhydrases. CONCLUSIONS CaNce103p is a β-class homotetrameric metalloenzyme composed of two homodimers. Its structure closely resembles those of other β-type carbonic anhydrases, in particular CAS1 from Sordaria macrospora. The main differences occur in the N-terminal part and the substrate tunnel. Detailed knowledge of the CaNce103p structure and the properties of the substrate tunnel in particular will facilitate design of selective inhibitors of this enzyme.

中文翻译：

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来自病原酵母白色念珠菌的碳酸酐酶CaNce103p的晶体结构。

背景技术由于碳酸酐酶CaNce103p，致病性酵母白色念珠菌可以在具有不同二氧化碳浓度的环境中增殖，这加速了二氧化碳自发转化为碳酸氢根，反之亦然。没有功能性CaNce103p，白色念珠菌无法在大气中生存。CaNce103p与来自酿酒酵母的直系同源物ScNce103p一起属于β-碳酸酐酶类。CaNce103p的晶体结构令人关注，因为该酶是表面消毒剂的潜在靶标。结果在大肠杆菌中制备了重组CaNce103p，并以2.2Å的分辨率确定了其晶体结构。CaNce103p形成组织为二聚体的二聚体的同四聚体，其中二聚体和四聚体表面垂直。尽管CaNce103p的生理作用与面包酵母的ScNce103p相似，但在结构水平上更类似于腐生真菌Sordaria macrospora的碳酸酐酶，后者也是四聚体。二聚化由亚基的N末端结构域中的两个螺旋介导。CaNce103p的N末端是柔性的，仅在前29个氨基酸被截短后才能获得晶体。对被29、48和61个氨基酸截断的CaNce103p变体的分析表明，残基30-48对于二聚化至关重要。每个亚基在活性位点均含有一个锌原子，并具有I型β-碳酸酐酶的特征。锌通过一个组氨酸残基，两个半胱氨酸残基和一个源自结晶缓冲液的β-巯基乙醇分子四面体配位。活性位点可通过底物通道进入，该通道比在其他真菌碳酸酐酶中观察到的长和窄。结论CaNce103p是由两个同型二聚体组成的β类同型四聚体金属酶。它的结构与其他β型碳酸酐酶的结构非常相似，特别是来自大红参（Sordaria macrospora）的CAS1。主要区别出现在N端部分和衬底隧道中。对CaNce103p结构和底物通道特性的详细了解将特别有助于设计该酶的选择性抑制剂。它的结构与其他β型碳酸酐酶的结构非常相似，特别是来自大红参（Sordaria macrospora）的CAS1。主要区别出现在N端部分和衬底隧道中。对CaNce103p结构和底物通道特性的详细了解将特别有助于设计该酶的选择性抑制剂。它的结构与其他β型碳酸酐酶的结构非常相似，特别是来自大红参（Sordaria macrospora）的CAS1。主要区别出现在N端部分和衬底隧道中。对CaNce103p结构和底物通道特性的详细了解将特别有助于设计该酶的选择性抑制剂。