Institute of Medical Technology, University of Tampere and Tampere University Hospital, Finland.
BMC Biochem. 2010 Jul 26;11:28. doi: 10.1186/1471-2091-11-28.
The beta-carbonic anhydrase (CA, EC 4.2.1.1) enzymes have been reported in a variety of organisms, but their existence in animals has been unclear. The purpose of the present study was to perform extensive sequence analysis to show that the beta-CAs are present in invertebrates and to clone and characterize a member of this enzyme family from a representative model organism of the animal kingdom, e.g., Drosophila melanogaster.
The novel beta-CA gene, here named DmBCA, was identified from FlyBase, and its orthologs were searched and reconstructed from sequence databases, confirming the presence of beta-CA sequences in 55 metazoan species. The corresponding recombinant enzyme was produced in Sf9 insect cells, purified, kinetically characterized, and its inhibition was investigated with a series of simple, inorganic anions. Holoenzyme molecular mass was defined by dynamic light scattering analysis and gel filtration, and the results suggested that the holoenzyme is a dimer. Double immunostaining confirmed predictions based on sequence analysis and localized DmBCA protein to mitochondria. The enzyme showed high CO2 hydratase activity, with a kcat of 9.5 x 105 s-1 and a kcat/KM of 1.1 x 108 M-1s-1. DmBCA was appreciably inhibited by the clinically-used sulfonamide acetazolamide, with an inhibition constant of 49 nM. It was moderately inhibited by halides, pseudohalides, hydrogen sulfide, bisulfite and sulfate (KI values of 0.67 - 1.36 mM) and more potently by sulfamide (KI of 0.15 mM). Bicarbonate, nitrate, nitrite and phenylarsonic/boronic acids were much weaker inhibitors (KIs of 26.9 - 43.7 mM).
The Drosophila beta-CA represents a highly active mitochondrial enzyme that is a potential model enzyme for anti-parasitic drug development.
β-碳酸酐酶（CA，EC 4.2.1.1）已在多种生物中被报道，但在动物中的存在尚不清楚。本研究的目的是进行广泛的序列分析，以证明β-CAs 存在于无脊椎动物中，并从动物王国的代表性模式生物果蝇中克隆和表征该酶家族的一个成员。
从 FlyBase 中鉴定出新型β-CA 基因，命名为 DmBCA，并从序列数据库中搜索和重建其同源物，确认了 55 种后生动物物种中β-CA 序列的存在。相应的重组酶在 Sf9 昆虫细胞中产生，进行了动力学表征，并研究了一系列简单的无机阴离子对其抑制作用。动态光散射分析和凝胶过滤确定了全酶的分子量，结果表明全酶是二聚体。双重免疫染色证实了基于序列分析的预测，并将 DmBCA 蛋白定位到线粒体。该酶具有高 CO2 水合酶活性，kcat 为 9.5 x 105 s-1，kcat/KM 为 1.1 x 108 M-1s-1。DmBCA 被临床使用的磺胺类药物乙酰唑胺显著抑制，抑制常数为 49 nM。它被卤化物、拟卤化物、硫化氢、亚硫酸氢盐和硫酸盐中度抑制（KI 值为 0.67 - 1.36 mM），被磺胺类药物更强烈抑制（KI 值为 0.15 mM）。碳酸氢盐、硝酸盐、亚硝酸盐和苯胂/硼酸是较弱的抑制剂（KI 值为 26.9 - 43.7 mM）。
果蝇β-CA 代表一种高度活跃的线粒体酶，可能成为抗寄生虫药物开发的模型酶。
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