Lee Y H, Ogata C, Pflugrath J W, Levitt D G, Sarma R, Banaszak L J, Pilkis S J
Department of Biochemistry, University of Minnesota, Minneapolis 55455, USA.
Biochemistry. 1996 May 14;35(19):6010-9. doi: 10.1021/bi9600613.
The crystal structure of the recombinant fructose-2,6-bisphosphatase domain, which covers the residues between 251 and 440 of the rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was determined by multiwavelength anomalous dispersion phasing and refined at 2.5 A resolution. The selenomethionine-substituted protein was induced in the methionine auxotroph, Escherichia coli DL41DE3, purified, and crystallized in a manner similar to that of the native protein. Phase information was calculated using the multiwavelength anomalous dispersion data collected at the X-ray wavelengths near the absorption edge of the K-shell alpha electrons of selenium. The fructose-2,6-bisphosphatase domain has a core alpha/beta structure which consists of six stacked beta-strands, four parallel and two antiparallel. The core beta-sheet is surrounded by nine alpha-helices. The catalytic site, as defined by a bound phosphate ion, is positioned near the C-terminal end of the beta-sheet and close to the N-terminal end of an alpha-helix. The active site pocket is funnel-shaped. The narrow opening of the funnel is wide enough for a water molecule to pass. The key catalytic residues, including His7, His141, and Glu76, are near each other at the active site and probably function as general acids and/or bases during a catalytic cycle. The inorganic phosphate molecule is bound to an anion trap formed by Arg6, His7, Arg56, and His141. The core structure of the Fru-2,6-P2ase is similar to that of the yeast phosphoglycerate mutase and the rat prostatic acid phosphatase. However, the structure of one of the loops near the active site is completely different from the other family members, perhaps reflecting functional differences and the nanomolar range affinity of Fru-2,6-P2ase for its substrate. The imidazole rings of the two key catalytic residues, His7 and His141, are not parallel as in the yeast phosphoglycerate mutase. The crystal structure is used to interpret the existing chemical data already available for the bisphosphatase domain. In addition, the crystal structure is compared with two other proteins that belong to the histidine phosphatase family.
重组果糖-2,6-二磷酸酶结构域的晶体结构已通过多波长反常色散相位测定法确定，并在2.5埃分辨率下进行了精修。该结构域覆盖大鼠肝脏双功能酶6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶251至440位的残基。硒代甲硫氨酸取代的蛋白质在甲硫氨酸营养缺陷型大肠杆菌DL41DE3中诱导表达、纯化，并以与天然蛋白质相似的方式结晶。利用在硒的K壳层α电子吸收边缘附近的X射线波长处收集的多波长反常色散数据计算相位信息。果糖-2,6-二磷酸酶结构域具有核心α/β结构，由六条堆叠的β链组成，其中四条平行，两条反平行。核心β折叠被九条α螺旋包围。由结合的磷酸根离子定义的催化位点位于β折叠的C末端附近，靠近一条α螺旋的N末端。活性位点口袋呈漏斗状。漏斗的狭窄开口宽到足以让一个水分子通过。关键的催化残基，包括His7、His141和Glu76，在活性位点彼此靠近，可能在催化循环中作为广义酸和/或碱发挥作用。无机磷酸分子与由Arg6、His7、Arg56和His141形成的阴离子陷阱结合。果糖-2,6-二磷酸酶的核心结构与酵母磷酸甘油酸变位酶和大鼠前列腺酸性磷酸酶的核心结构相似。然而，活性位点附近一个环的结构与其他家族成员完全不同，这可能反映了功能差异以及果糖-2,6-二磷酸酶对其底物的纳摩尔范围亲和力。两个关键催化残基His7和His141的咪唑环不像酵母磷酸甘油酸变位酶那样平行。晶体结构用于解释已有的关于双磷酸酶结构域的化学数据。此外，将该晶体结构与另外两种属于组氨酸磷酸酶家族的蛋白质进行了比较。
