Crystal structure of a pyrimidine dimer-specific excision repair enzyme from bacteriophage T4: refinement at 1.45 A and X-ray analysis of the three active site mutants.

摘要: Crystallographic study of bacteriophage T4 endonuclease V, which is involved in the initial step pyrimidine dimer-specific excision repair pathway, has been carried out with respect to wild-type and three different mutant enzymes. This enzyme catalyzes cleavage N-glycosyl bond at 5'-side dimer, subsequently incises phosphodiester apyrimidinic site through a beta-elimination reaction. The structure refined 1.45 A resolution reveals detailed molecular architecture. composed single compact domain classified as an all-alpha structure. molecule stabilized mainly by hydrophobic cores, two include many aromatic side-chain interactions. unique folding motif, where amino-terminal segment penetrates between major alpha-helices prevents their direct contact, it incompatible close-packing category helices for protein folding. concave surface, covered positive charges, implies interface DNA binding. glycosylase catalytic center, comprises Glu23 surrounding basic residues Arg3, Arg22 Arg26, lie this surface. crystal structures active-site mutants, was replaced Gln(E23Q) Asp (E23D), respectively, Arg3 Gln (R3Q), have determined atomic resolution. backbone E23Q R3Q mutants were almost identical that wild-type, while E23D mutation induces small, but significant, change structure, such increase central kink H1 helix Pro25. In center glycosylase, however, these mutations do not generate notable movements atoms, except significant shifts some bound water molecules. Thus, structural differences each are confined remarkably small region around chemical groups. Combined biochemical studies difference circular dichroism measurements, results allow us conclude negatively charged carboxyl group essential bond, positively guanidino crucial bind substrate, duplex containing dimer. amino terminal alpha-amino located position approximately 4.4 away from Glu23. These features generally consistent reaction scheme proposed Dodson co-workers.