Thomson G J, Howlett G J, Ashcroft A E, Berry A
Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K.
Biochem J. 1998 Apr 15;331 ( Pt 2)(Pt 2):437-45. doi: 10.1042/bj3310437.
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 GenBanktrade mark database [gene dhnA; entry code U73760; Close and Choi (1996) Submission to GenBanktrade mark]. 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 (Lys236) has been identified by a combination of site-directed mutagenesis, kinetics and electrospray-ionization MS. A second lysine residue (Lys238) has been implicated in substrate binding. The cloning of this gene and the high levels of overexpression obtained will facilitate future structure-function studies.
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，并且该蛋白质已大量过量表达。此基因序列先前在GenBank商标数据库中被鉴定为编码一种大肠杆菌脱水蛋白[基因dhnA；登录号U73760；克洛斯和崔（1996年）提交至GenBank商标数据库]。然而，从dhnA基因过量表达纯化得到的蛋白质具有大肠杆菌I类FBP醛缩酶所有已知的特性。该蛋白质是一种8至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类酶与其他已知的FBP醛缩酶（包括I类和II类）的序列同一性较低（约为20%），这可能反映在这种FBP醛缩酶的一些新特性上。活性位点肽已被分离出来，并且通过定点诱变、动力学和电喷雾电离质谱相结合的方法鉴定出了形成席夫碱的赖氨酸残基（Lys236）。第二个赖氨酸残基（Lys238）与底物结合有关。该基因的克隆以及所获得的高水平过量表达将有助于未来的结构-功能研究。
