title: Dynamic roles of arginine residues 82 and 92 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: crystallographic studies.
abstract: 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), the first reaction in the folate biosynthetic pathway. Arginine residues 82 and 92, strictly conserved in 35 HPPK sequences, play dynamic roles in the catalytic cycle of the enzyme. At 0.89-A resolution, two distinct conformations are observed for each of the two residues in the crystal structure of the wild-type HPPK in complex with two HP variants, two Mg(2+) ions, and an ATP analogue. Structural information suggests that R92 first binds to the alpha-phosphate group of ATP and then shifts to interact with the beta-phosphate as R82, which initially does not bind to ATP, moves in and binds to alpha-phosphate when the pyrophosphoryl transfer is about to occur. The dynamic roles of R82 and R92 are further elucidated by five more crystal structures of two mutant proteins, R82A and R92A, with and without bound ligands. Two oxidized forms of HP are observed with an occupancy ratio of 0.50:0.50 in the 0.89-A structure. The oxidation of HP has significant impact on its binding to the protein as well as the conformation of nearby residue W89.
authors.affiliation: Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, Maryland 21702, USA.
authors.forename: Jaroslaw
authors.initials: J
authors.lastname: Blaszczyk
authors.forename: Yue
authors.initials: Y
authors.lastname: Li
authors.forename: Genbin
authors.initials: G
authors.lastname: Shi
authors.forename: Honggao
authors.initials: H
authors.lastname: Yan
authors.forename: Xinhua
authors.initials: X
authors.lastname: Ji
mesh_terms: D000409:Alanine / Q000235:genetics; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002413:Cations, Divalent; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D017854:Diphosphotransferases / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D008274:Magnesium / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017354:Point Mutation; D011487:Protein Conformation; D011621:Pteridines / Q000737:chemistry; D011622:Pterins; D021281:Quantitative Structure-Activity Relationship
chemical_list: D002413:Cations, Divalent; D029968:Escherichia coli Proteins; D011621:Pteridines; D011622:Pterins; C025596:7,8-dihydropterin; D001120:Arginine; D017854:Diphosphotransferases; C019684:2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase; D008274:Magnesium; D000409:Alanine