Dostál Jiří, Brynda Jiří, Blaha Jan, Macháček Stanislav, Heidingsfeld Olga, Pichová Iva
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo náměstí 2, 166 10, Prague, Czech Republic.
Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Studentská 95, 532 10, Pardubice, Czech Republic.
BMC Struct Biol. 2018 Oct 26;18(1):14. doi: 10.1186/s12900-018-0093-4.
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.
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.
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.
致病性酵母白色念珠菌能够在不同二氧化碳浓度的环境中增殖，这要归功于碳酸酐酶CaNce103p，它能加速二氧化碳与碳酸氢盐之间的自发转化，反之亦然。没有功能性的CaNce103p，白色念珠菌无法在大气环境中存活。CaNce103p与酿酒酵母的直系同源蛋白ScNce103p同属于β-碳酸酐酶类。CaNce103p的晶体结构备受关注，因为这种酶是表面消毒剂的潜在作用靶点。
在大肠杆菌中制备了重组CaNce103p，并以2.2 Å的分辨率测定了其晶体结构。CaNce103p形成一个同四聚体，由两个二聚体组成二聚体的形式，其中二聚化表面和四聚化表面相互垂直。虽然CaNce103p的生理作用与面包酵母中的ScNce103p相似，但在结构层面上，它更类似于腐生真菌大孢粪壳菌的碳酸酐酶，后者也是四聚体。二聚化由亚基N端结构域中的两个螺旋介导。CaNce103p的N端是灵活的，只有在前29个氨基酸被截短后才获得晶体。对截短了29、48和61个氨基酸的CaNce103p变体的分析表明，第30 - 48位氨基酸对于二聚化至关重要。每个亚基在活性位点含有一个锌原子，并表现出I型β-碳酸酐酶的特征。锌由一个组氨酸残基、两个半胱氨酸残基和一个来自结晶缓冲液的β-巯基乙醇分子以四面体方式配位。活性位点可通过底物通道进入，这些通道比在其他真菌碳酸酐酶中观察到的通道略长且略窄。
CaNce103p是一种由两个同二聚体组成的β类同四聚体金属酶。其结构与其他β型碳酸酐酶的结构非常相似，特别是大孢粪壳菌的CAS1。主要差异出现在N端部分和底物通道。特别是对CaNce103p结构和底物通道特性的详细了解将有助于设计这种酶的选择性抑制剂。
