Oberholzer Anselm Erich, Schneider Philipp, Baumann Ulrich, Erni Bernhard
Departement of Chemistry und Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Bern, Switzerland.
J Mol Biol. 2006 Jun 9;359(3):539-45. doi: 10.1016/j.jmb.2006.03.057. Epub 2006 Apr 18.
Dihydroxyacetone (Dha) kinases are a family of sequence-related enzymes that utilize either ATP or phosphoenolpyruvate (PEP) as source of high energy phosphate. The PEP-dependent Dha kinase of Escherichia coli consists of three subunits. DhaK and DhaL are homologous to the Dha and nucleotide-binding domains of the ATP-dependent kinase of Citrobacter freundii. The DhaM subunit is a multiphosphorylprotein of the PEP:sugar phosphotransferase system (PTS). DhaL contains a tightly bound ADP as coenzyme that gets transiently phosphorylated in the double displacement of phosphate between DhaM and Dha. Here we report the 2.6A crystal structure of the E.coli DhaL subunit. DhaL folds into an eight-helix barrel of regular up-down topology with a hydrophobic core made up of eight interlocked aromatic residues and a molecule of ADP bound at the narrower end of the barrel. The alpha and beta phosphates of ADP are complexed by two Mg2+ and by a hydrogen bond to the imidazole ring of an invariant histidine. The Mg ions in turn are coordinated by three gamma-carboxyl groups of invariant aspartate residues. Water molecules complete the octahedral coordination sphere. The nucleotide is capped by an alpha-helical segment connecting helices 7 and 8 of the barrel. DhaL and the nucleotide-binding domain of the C.freundii kinase assume the same fold but display strongly different surface potentials. The latter observation and biochemical data indicate that the domains of the C.freundii Dha kinase constitute one cooperative unit and are not randomly interacting and independent like the subunits of the E.coli enzyme.
二羟基丙酮（Dha）激酶是一类序列相关的酶，它们利用ATP或磷酸烯醇丙酮酸（PEP）作为高能磷酸源。大肠杆菌中依赖PEP的Dha激酶由三个亚基组成。DhaK和DhaL与弗氏柠檬酸杆菌中依赖ATP的激酶的Dha和核苷酸结合结构域同源。DhaM亚基是磷酸烯醇丙酮酸：糖磷酸转移酶系统（PTS）的多磷酸化蛋白。DhaL含有紧密结合的ADP作为辅酶，在DhaM和Dha之间的磷酸双位移过程中会短暂磷酸化。在此，我们报道了大肠杆菌DhaL亚基的2.6埃晶体结构。DhaL折叠成一个具有规则上下拓扑结构的八螺旋桶，其疏水核心由八个互锁的芳香族残基组成，并且在桶较窄的一端结合有一个ADP分子。ADP的α和β磷酸与两个Mg2+络合，并通过氢键与一个不变组氨酸的咪唑环相连。Mg离子又由不变天冬氨酸残基的三个γ-羧基配位。水分子完成八面体配位球。核苷酸由连接桶的螺旋7和8的α-螺旋片段封闭。DhaL和弗氏柠檬酸杆菌激酶的核苷酸结合结构域具有相同的折叠，但显示出截然不同的表面电位。后一观察结果和生化数据表明，弗氏柠檬酸杆菌Dha激酶的结构域构成一个协同单元，不像大肠杆菌酶的亚基那样随机相互作用且相互独立。
