Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases

Abstract

The 2.15-Å resolution cocrystal structure of EcoRV endonuclease mutant T93A complexed with DNA and Ca 2؉ ions reveals two divalent metals bound in one of the active sites. One of these metals is ligated through an innersphere water molecule to the phosphate group located 3 to the scissile phosphate. A second inner-sphere water on this metal is positioned approximately in-line for attack on the scissile phosphate. This structure corroborates the observation that the pro-S P phosphoryl oxygen on the adjacent 3 phosphate cannot be modified without severe loss of catalytic efficiency. The structural equivalence of key groups, conserved in the active sites of EcoRV, EcoRI, PvuII, and BamHI endonucleases, suggests that ligation of a catalytic divalent metal ion to this phosphate may occur in many type II restriction enzymes. Together with previous cocrystal structures, these data allow construction of a detailed model for the pretransition state configuration in EcoRV. This model features three divalent metal ions per active site and invokes assistance in the bond-making step by a conserved lysine, which stabilizes the attacking hydroxide ion nucleophile.Recently determined crystal structures of type II restriction endonucleases have produced a wealth of information on the basis for target site sequence selectivity (1-6). However, these structures do not resolve the detailed structural mechanism of catalysis. Although acidic and basic groups in the active sites can be identified, and in some cases divalent-metal binding sites delineated, a convincing picture clarifying the way in which the attacking hydroxide ion is generated, and the leaving group stabilized, has not been elucidated for any of the enzymes.EcoRV endonuclease is a homodimer of 244 aa per monomer. It cleaves the duplex sequence 5Ј-GATATC at the central TA step in a blunt-ended fashion (7). As in all type II enzymes, phosphoryl transfer proceeds via attack of a hydroxide ion nucleophile on the scissile phosphorus, generating products containing a 5Ј phosphate group. This reaction occurs by in-line displacement through a pentacovalent transition state, with inversion of stereochemistry at phosphorus and an absolute requirement for divalent metal cations (8, 9). Highresolution crystallographic analyses of EcoRV show that the scissile phosphates of the DNA are located adjacent to the carboxylates of Asp-90 and Asp-74, and that a divalent metal ion bridges the enzyme and DNA at this position (2, 3). Although this metal could stabilize the additional negative charge that develops in the transition state, it is not correctly positioned to generate the attacking hydroxide ion. Thus, speculative mechanisms in which the nucleophile arises from dissociation of a metal-ligated water have invoked significant rearrangements of the DNA from its observed conformation in the crystal structures (2, 10).An alternative mechanism involving substrate-assisted catalysis by the adjacent 3Ј-phosphate of the DNA also has been proposed (11,12). In support of this mechanis...

Crystallographic snapshots along a protein-induced DNA-bending pathway

“…3D) (25). Complete assembly of the active site requires accurate juxtaposition of Asp-90͞Lys-92 in the central ␤-sheet with Asp-74 and Glu-45, for ligation of required divalent cations that bind between these side chains and the DNA phosphates (25)(26)(27). A lattice contact on the amino-terminal helix of the enzyme, present in all crystals except the active form I, appears to prevent the B-helix movements (Fig.…”

Investigating HIV-1 Polypurine Tract Geometry via Targeted Insertion of Abasic Lesions in the (–)-DNA Template and (+)-RNA Primer

“…We therefore interpret loss of PPT/U3 cleavage with substrate Ϫ1RAb as loss or alteration of this critical interaction. With respect to the nucleobase 3Ј of the PPT/U3 junction, Haruki et al (35) have exploited RNA/DNA hybrids containing phosphorothioate substitutions to propose that the pro-R p -oxygen of the phosphate group 3Ј to the scissile bond cooperates during catalysis with the catalytic His-124 of E. coli RNase H, analogous to the model of substrate-assisted catalysis proposed for type II restriction endonucleases (36). Fig.…”

Enzyme-like activities of algal polysaccharide - cerium complexes

“…By the attacking of a hydroxide ion nucleophile on the position, which has invoked significant rearrangements of the DNA, chloropyrifos and pNPP, the ion Ce 4+ hydrolyzes the substances (Horton et aL, 1998).…”