CRYSTAL-STRUCTURE OF THE RAT-LIVER FRUCTOSE-2,6-BISPHOSPHATASE BASED ON SELENOMETHIONINE MULTIWAVELENGTH ANOMALOUS-DISPERSION PHASES

The crystal structure of the recombinant fructose-2,6-bisphosphatase d omain, which covers the residues between 251 and 440 of the rat liver bifunctional enzyme, phosphofructo-2-kinase/fructose2,6-bisphosphatase , was determined by multiwavelength anomalous dispersion phasing and r efined at 2.5 Angstrom resolution. The selenomethionine-substituted pr otein was induced in the methionine auxotroph, Escherichia coli DL41DE 3, purified, and crystallized in a manner similar to that of the nativ e 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 fructo se-2,6-bisphosphatase domain has a core alpha/beta structure which con sists of six stacked beta-strands, four parallel and two antiparallel. The core beta-sheet is surrounded by nine alpha-helices. The catalyti c 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 a lpha-helix. The active site pocket is funnel-shaped. The narrow openin g of the funnel is wide enough for a water molecule to pass, The key c atalytic residues, including His7, His141, and Glu76, are near each ot her at the active site and probably function as general acids and/or b ases during a catalytic cycle. The inorganic phosphate molecule is bou nd to an anion trap formed by Arg6, His7, Arg56, and Hisl41. The core structure of the Fru-2,6-P(2)ase is similar to that of the yeast phosp hoglycerate mutase and the rat prostatic acid phosphatase. However, th e structure of one of the loops near the active site is completely dif ferent from the other family members, perhaps reflecting functional di fferences and the nanomolar range affinity of Fru-2,6-P(2)ase for its substrate. The imidazole rings of the two key catalytic residues, His7 and Hisl41, 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 cry stal structure is compared with two other proteins that belong to the histidine phosphatase family.