Crystal structure and kinetic studies of a tetrameric type II β-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae
Crystal structure and kinetic studies of a tetrameric type II β-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae
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DOI：
10.1107/s1399004715018635
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发表时间：
2015-12-01
期刊：
影响因子：
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通讯作者：
Supuran, Claudiu T.
中科院分区：
文献类型：
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作者：
Ferraroni, Marta;Del Prete, Sonia;Supuran, Claudiu T.
碳酸酐酶（CA）是一种锌酶，催化二氧化碳可逆转化为碳酸氢盐（碳酸氢盐）和质子。CA由于参与了许多生理和病理过程而被广泛研究。目前，CA抑制剂被广泛用作抗青光眼、抗癌和抗肥胖药物以及用于治疗神经系统疾病。近年来，利用CA抑制剂对抗由原生动物、真菌和细菌引起的感染已成为一个新的研究方向。本文报道了霍乱弧菌β-CA（VchCA β）的克隆及其动力学特性。这种新酶的X射线晶体结构在1.9埃分辨率下从完美的部分面体孪生晶体中解析出来，揭示了具有封闭活性位点的四聚体II型β-CA，其中锌与Cys 42，Asp 44，His 98和Cys 101四面体配位。发现底物碳酸氢盐结合在靠近锌离子的非催化结合口袋中，正如其他一些β-CA所报告的那样，例如来自大肠杆菌和流感嗜血杆菌的β-CA。在pH 8.3时，该酶对CO2水合为碳酸氢根和质子的生理反应表现出显著的催化活性，动力学参数如下：kcat = 3.34 x 10（5）s（-1），k（cat）/K-m = 4.1 x 10（7）M-1 s（-1）。另一方面，乙酰唑胺（Ki为4.5 μ M）对新酶的抑制作用较差。由于这种细菌病原体编码至少三种CA，α-CA、β-CA和γ-CA，这些酶可能在弧菌的生命周期和致病性中起重要作用，并且不能排除可以在治疗上利用对其活性的干扰来获得具有不同作用机制的抗生素。
Carbonic anhydrase (CA) is a zinc enzyme that catalyzes the reversible conversion of carbon dioxide to bicarbonate (hydrogen carbonate) and a proton. CAs have been extensively investigated owing to their involvement in numerous physiological and pathological processes. Currently, CA inhibitors are widely used as antiglaucoma, anticancer and anti-obesity drugs and for the treatment of neurological disorders. Recently, the potential use of CA inhibitors to fight infections caused by protozoa, fungi and bacteria has emerged as a new research direction. In this article, the cloning and kinetic characterization of the beta-CA from Vibrio cholerae (VchCA beta) are reported. The X-ray crystal structure of this new enzyme was solved at 1.9 angstrom resolution from a crystal that was perfectly merohedrally twinned, revealing a tetrameric type II beta-CA with a closed active site in which the zinc is tetrahedrally coordinated to Cys42, Asp44, His98 and Cys101. The substrate bicarbonate was found bound in a noncatalytic binding pocket close to the zinc ion, as reported for a few other beta-CAs, such as those from Escherichia coli and Haemophilus influenzae. At pH 8.3, the enzyme showed a significant catalytic activity for the physiological reaction of the hydration of CO2 to bicarbonate and protons, with the following kinetic parameters: a kcat of 3.34 x 10(5) s(-1) and a k(cat)/K-m of 4.1 x 10(7) M-1 s(-1). The new enzyme, on the other hand, was poorly inhibited by acetazolamide (K-i of 4.5 mu M). As this bacterial pathogen encodes at least three CAs, an alpha-CA, a beta-CA and a gamma-CA, these enzymes probably play an important role in the life cycle and pathogenicity of Vibrio, and it cannot be excluded that interference with their activity may be exploited therapeutically to obtain antibiotics with a different mechanism of action.