A CARBONIC-ANHYDRASE FROM THE ARCHAEON METHANOSARCINA-THERMOPHILA
A CARBONIC-ANHYDRASE FROM THE ARCHAEON METHANOSARCINA-THERMOPHILA
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DOI：
10.1073/pnas.91.15.6909
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发表时间：
1994-07-19
影响因子：
11.1
通讯作者：
FERRY, JG
中科院分区：
文献类型：
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作者：
ALBER, BE;FERRY, JG
从醋酸盐生长的嗜热甲烷单胞菌中分离纯化了碳酸酐酶(CA)，纯化倍数为10,000倍(回收率为22%)，比活力为4872单位/毫克。凝胶过滤层析测得该酶的天然相对分子质量为84 kDa。SDS/PAGE显示一条表观分子质量为40 kDa的蛋白条带。嗜热支原体CA对磺胺类药物和单价离子的抑制作用不如人CA同工酶II敏感。将编码该CA的基因克隆到pUC18中，并进行了序列测定。表达产物的氨基酸序列与纯化蛋白的N-端序列比较表明，该基因编码34个N-末端残基，具有分泌蛋白信号肽的特性。计算的相对分子质量(22.9 kDa)和等电点(4.0)表明，SDS/PAGE高估了提交大小，说明天然酶是四聚体。据我们所知，推导的氨基酸序列与任何已知的CA没有显著的同源性，但与聚球藻PCC7942提出的二氧化碳浓缩机制蛋白的前197个氨基酸有35%的同源性，与铜绿假单胞菌铁蛋白结合蛋白的推导序列有28%的同源性。因此，我们的结果表明，这种古老的CA代表了一个不同的CA类别，并为确定CA在乙酰营养型厌氧菌中的生理作用提供了基础。
Carbonic anhydrase (CA) from acetate-grown Methanosarcina thermophila was purified >10,000-fold (22% recovery) to apparent homogeneity with a specific activity of 4872 units/mg. The estimated native molecular mass of the enzyme is 84 kDa based on gel filtration chromatography. SDS/PAGE revealed one protein band with an apparent molecular mass of 40 kDa. The M. thermophila CA is less sensitive than human CA isozyme II toward inhibition by sulfonamides and monovalent ions. The gene encoding this CA was cloned into pUC18 and sequenced. Escherichia coli harboring the recombinant plasmid expresses CA activity (2.3 units/mg of cell extract protein), Comparison of the deduced amino acid sequence with the N-terminal sequence of the purified protein shows that the gene encodes an additional 34 N-terminal residues with properties characteristic of signal peptides in secretory proteins. The calculated molecular mass (22.9 kDa) and pI (4.0) suggest that SDS/PAGE overestimates the submit size and that the native enzyme is a tetramer. To our knowledge, the deduced amino acid sequence has no significant identity to any known CA but has 35% sequence identity to the first 197 deduced N-terminal amino acids of a proposed CO2-concentrating-mechanism protein from Synechococcus PCC7942 and 28% sequence identity to the deduced sequence of ferripyochelin binding protein from Pseudomonas aeruginosa. Thus, our results indicate that this archaeal CA represents a distinct class of CAs and provide a basis to determine physiological roles for CA in acetotrophic anaerobes.