Mechanism of DNA Cleavage

The kinetics and mechanism of catalysis have been particularly well studied for the I-Crel enzyme (Chevalier et al. 2004); many of these results appear to be generalizable to the LAGLIDADG family. The measured single-turnover kinetic rate constants, k* „ and K * of the wild-type I-Crel enzyme are roaX ro • •

0.03 min l and l.OxlO-4 nM, respectively, giving a value for catalytic efficiency (k*max/Km*) of 0.3 nM_1min_1. This enzyme and its relatives are all dependent on divalent cations for activity, similar to most if not all known endonucleas-es. A wide variety of divalent metal ions have been assayed for cleavage activity with I-Crel and display a wide range of effects (Chevalier et al. 2004). Two metals (calcium and copper) fail to support cleavage, two (nickel and zinc) display reduced cleavage activity, and three (magnesium, cobalt and manganese) display full activity under the conditions tested. The use of manganese in place of magnesium allows recognition and cleavage of a broader repertoire of DNA target sequences than is observed with magnesium, as is seen for a variety of endonuclease catalysts such as restriction enzymes.

The structures of the four endonuclease-DNA complexes that have been solved at relatively high resolution (I-Crel, I-Msol, I-Scel and H-Drel) all indicate the presence of three bound divalent metal ions coordinated by a pair of overlapping active sites, with one shared metal participating in both cleavage reactions by virtue of interacting with the scissile phosphates and 3' hydroxyl leaving groups on both DNA strands. The structures of these four enzymes differ somewhat in the precise position and binding interactions of the metals, but point to similar mechanisms where each strand is cleaved using a canonical two-metal mechanism for phosphodiester hydrolysis (Fig. 4). Whether this

Canonical Splicing

Fig. 4. Proposed mechanism of DNA hydrolysis for the I-Crel homing endonuclease. Other LAGLIDADG enzymes are also thought to follow a canonical two-metal phosphoryl hydrolysis pathway, but with significant variation in the positions and/or roles of basic residues and ordered water molecules. Those residues shown are all known to be essential or extremely important for DNA cleavage by I-Crel. Other LAGLIDADG enzymes display significant divergence at all positions except for the direct metal-binding residues (Asp 20 and 20') from the LAGLIDADG motifs (Table 1)

Fig. 4. Proposed mechanism of DNA hydrolysis for the I-Crel homing endonuclease. Other LAGLIDADG enzymes are also thought to follow a canonical two-metal phosphoryl hydrolysis pathway, but with significant variation in the positions and/or roles of basic residues and ordered water molecules. Those residues shown are all known to be essential or extremely important for DNA cleavage by I-Crel. Other LAGLIDADG enzymes display significant divergence at all positions except for the direct metal-binding residues (Asp 20 and 20') from the LAGLIDADG motifs (Table 1)

unusual structural feature - a shared central divalent metal ion - imparts any particular kinetic order (or simultaneity) to the individual cleavage events is not known for the homodimeric enzymes. In contrast, the structure of the asymmetric I-Scel-DNA complex (Moure et al. 2003) clearly demonstrates that DNA cleavage must involve sequential cleavage of coding and non-coding DNA strands, with a significant conformational rearrangement of the active sites relative to DNA occurring between the two reactions.

In contrast, the structures of DNA complexes of one monomeric enzyme (I-Anil; Bolduc et al. 2003) and of the intein-associated Pl-Scel, solved at lower resolution (~3 A; Moure et al. 2002), have thus far revealed the presence of only two bound metal ions; a central, shared metal ion is not visible. It is unclear whether this reflects a significant difference in catalytic mechanism, reduced occupancy or poor structural ordering of the central metal ion, or simply a limitation of lower resolution crystallographic data.

In the high-resolution structures listed above, a single independently bound metal in each of the two endonuclease active sites coordinates a directly ligated water molecule, which is appropriately positioned for an in-line hy-drolytic attack on a scissile phosphate group. The third, 'shared' central metal ion stabilizes the transition state phospho anion and the 3' hydroxylate leaving group for both strand cleavage events (Chevalier et al. 2001). In I-Crel, the central metal is jointly coordinated by one conserved acidic residue from each LAGLIDADG motif and by oxygen atoms from the scissile phosphates of each DNA strand. The unshared metals in each individual active site are also coordinated by a single LAGLIDADG carboxylate oxygen, as well as a non-bridging DNA oxygen atom and a well-ordered coordination shell of water molecules. One of the metal-bound water molecules in the active site is often in contact with a catalytically essential glutamine or asparagine residue. In addition to the attacking water molecule a well-ordered network of water molecules is distributed in a large pocket surrounding the DNA scissile phosphate group. These ordered solvent molecules extend from the metal-bound nucle-ophile to the leaving group 3' oxygen and are themselves positioned or coordinated by several basic residues that line the solvent pocket.

At physiological pH, phosphate ester bonds have large barriers to cleavage even though they are thermodynamically unstable (Westheimer 1987). To efficiently catalyze the cleavage of phosphate esters, several chemical features are required, including a nucleophile, a basic moiety to activate and position that nucleophile, a general acid to protonate the leaving group, and the presence of one or more positively charged groups to stabilize the phosphoanion transition state (Galburt and Stoddard 2002). The diversity of chemical groups and metal ions available to proteins has made it possible for evolution to arrive at many diverse strategies that satisfy the above requirements. A common feature of many endonucleases (and other phosphoryl transfer enzymes) is the use of bound metal ions as cofactors, and a basic residue (such as a lysine) that directly activates the water molecule for nucleophilic attack.

The metal-dependent features of DNA hydrolysis described above are clearly imparted in the LAGLIDADG endonucleases by the conserved acidic residues of their namesake sequence motif, which directly coordinate divalent cations. However, the remaining residues in the active site are remarkable for their chemical and structural diversity (Chevalier and Stoddard 2001; Table 1). In fact, no enzyme in this family has an essential residue that has been unambiguously identified as a general base for activation of a water nucle-ophile. Indeed, these enzymes are unique compared to other hydrolytic endonucleases in that the basic residues in their active sites are not generally found in contact distance with metal-bound waters. Catalytically important basic residues, such as Lys 98 in I-Crel, which are involved in interactions with solvent molecules (including those in contact with the scissile phosphate), are poorly conserved, and in some cases absent. The only obvious common chemical feature of many of those residues is the capacity to either donate or accept one or more hydrogen bonds. It is possible that these peripheral active site residues are responsible for positioning and polarizing the solvent network in the active site to facilitate efficient proton transfer reactions to and from nucleophiles and 3' leaving groups. Each branch of closely related enzymes may have adopted a unique active site solvent packing arrangement that is highly specialized. Furthermore, this rapidly diverging enzyme family

Table 1. Summary of conserved motif and active site residues for LAGLIDADG homing endonuclease structures

Enzyme

LAGLIDADG

Metal Binding

Basic Pocket

I-Crel

LAGFVDGDG

D20

Q47

K98

R51

I-Msol

IAGFLDGDG

D21

Q49

K104

K54

I-Dmol

LLGLIIGDG

D21

Q42

K120

K43

IKGLYVAEG

E117

N129

-

K130

I-Anil

LVGLFEGDG

D15

L36

K94

D40

LVGFIEAEG

E148

Q171

K227

G174

I-Scel

GIGLILGDA

D44

E61

K122

-

LAYWFMDDG

D145

N192

K223

-

PI-SceI

LLGLWIGDG

D218

D229

K301

R231

LAGLIDSDG

D326

T341

K403

H343

PI-PfuI

LAGFIAGDG

D149

D173

L220

-

IAGLFDAEG

E250

M263

K322

-

may be broadly sampling and adopting significantly different combinations and configurations of chemical groups and associated water molecules to fulfill the catalytic roles described above.

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