The dhnA gene of Escherichia coli encodes a Class I fructose bisphosphate aldolase
The dhnA gene of Escherichia coli encodes a Class I fructose bisphosphate aldolase
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DOI：
10.1042/bj3310437
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发表时间：
1998-04-15
影响因子：
4.1
通讯作者：
Berry, A
中科院分区：
文献类型：
--
作者：
Thomson, GJ;Howlett, GJ;Berry, A
编码大肠杆菌I类果糖-1，6-二磷酸醛缩酶（FBP醛缩酶）的基因已被克隆，该蛋白质大量过量生产。该基因序列先前已被鉴定为编码E. GenBank（TM）数据库中的大肠杆菌然而，从dhnA基因过量产生的纯化蛋白与E. coli Class I FBP醛缩酶。该蛋白质是一个8-10聚体，天然分子量约为。340 kDa，每个亚基由349个氨基酸组成。I类酶显示出与其它已知的FBP醛缩酶（I类和II类）的低序列同一性（约20%），这可以通过该FBP醛缩酶的一些新性质来反映。活性位点肽已被分离，并已通过定点诱变，动力学和电喷雾电离MS的组合确定了席夫碱形成赖氨酸残基（Lys（236））。第二个赖氨酸残基（Lys（238））已被牵连在底物结合。该基因的克隆和获得的高水平过表达将有助于未来的结构功能研究。
The gene encoding the Escherichia coli Class I fructose-1,6-bisphosphate aldolase (FBP aldolase) has been cloned and the protein overproduced in high amounts. This gene sequence has previously been identified as encoding an E. coli dehydrin in the GenBank(TM) database [gene dhnA; entry code U73760; Close and Choi (1996) Submission to GenBank(TM)]. However, the purified protein overproduced from the dhnA gene shares all its properties with those known for the E. coli Class I FBP aldolase. The protein is an 8-10-mer with a native molecular mass of approx. 340 kDa, each subunit consisting of 349 amino acids. The Class I enzyme shows low sequence identity with other known FBP aldolases, both Class I and Class II (in the order of 20%), which may be reflected by some novel properties of this FBP aldolase. The active-site peptide has been isolated and the Schiff-base-forming lysine residue (Lys(236)) has been identified by a combination of site-directed mutagenesis, kinetics and electrospray-ionization MS. A second lysine residue (Lys(238)) has been implicated in substrate binding. The cloning of this gene and the high levels of overexpression obtained will facilitate future structure function studies.