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J. Biol. Chem., Vol. 280, Issue 30, 27990-27997, July 29, 2005

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Structural Insights into the Mechanism of Nuclease A, a Metal Nuclease from Anabaena^*

Mahua Ghosh, Gregor Meiss, Alfred Pingoud, Robert E. London¶, and Lars C. Pedersen

From the Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 and the Institut für Biochemie, FB08, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany

Received for publication, February 17, 2005 , and in revised form, May 12, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
Nuclease A (NucA) is a nonspecific endonuclease from Anabaena sp. capable of degrading single- and double-stranded DNA and RNA in the presence of divalent metal ions. We have determined the structure of the (2-24),D121A mutant of NucA in the presence of Zn²⁺ and Mn²⁺ (PDB code 1ZM8 [PDB] ). The mutations were introduced to remove the N-terminal signal peptide and to reduce the activity of the nonspecific nuclease, thereby reducing its toxicity to the Escherichia coli expression system. NucA contains a metal finger motif and a hydrated Mn²⁺ ion at the active site. Unexpectedly, NucA was found to contain additional metal binding sites 26 Å apart from the catalytic metal binding site. A structural comparison between NucA and the closest analog for which structural data exist, the Serratia nuclease, indicates several interesting differences. First, NucA is a monomer rather than a dimer. Second, there is an unexpected structural homology between the N-terminal segments despite a poorly conserved sequence, which in Serratia includes a cysteine bridge thought to play a regulatory role. In addition, although a sequence alignment had suggested that NucA lacks a proposed catalytic residue corresponding to Arg⁵⁷ in Serratia, the structure determined here indicates that Arg⁹³ in NucA is positioned to fulfill this role. Based on comparison with DNA-bound nuclease structures of the metal finger nuclease family and available mutational data on NucA, we propose that His¹²⁴ acts as a catalytic base, and Arg⁹³ participates in the catalysis possibly through stabilization of the transition state.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
Nuclease A (NucA)¹ from Anabaena sp. PCC 7120 (1) is a member of a family of highly active, divalent metal ion-dependent, nonspecific nucleases, which are characterized by the DRGH prosite motif. NucA is able to degrade both single- and double-stranded DNA and RNA and functions optimally in the presence of Mn²⁺ or Mg²⁺ ions (2). The extracellular nuclease from Serratia marcescens (3), yeast Nuc1 (4), mitochondrial EndoG (5), and Streptococcus pneumoniae EndA (6) are some of the other family members. The best characterized of these is the Serratia nuclease (7), a homodimer (8-11) that contains two disulfide bonds per subunit (12) and is characterized by an unusual Mg²⁺ binding site optimized for binding a hydrated metal ion (13, 14).

The prokaryotic nucleases of this family generally contain an N-terminal signal peptide and are secreted into the extracellular environment (15). It is believed that most of them serve nutritional purposes and possibly also function as bacteriocides, similar to the colicins of Escherichia coli (16). Because nonspecific nucleases will generally be extremely toxic to the cells that produce them, various mechanisms have evolved to deal with their toxicity. Many of the nucleases in this class contain disulfide bonds that activate the nucleases upon secretion into an oxidizing environment but when reduced in the intracellular environment render the enzyme inactive (15). If the cysteine residues in the N-terminal signal peptide are not included, there is only one additional cysteine in NucA, and hence it is not anticipated to contain disulfide bonds, which could prevent the nuclease from being active in the cell. Instead, NucA is paired with a specific inhibitor, NuiA, analogous to the colicin immunity proteins in E. coli (17). NuiA protects the cell from nuclease action by forming a very stable NucA·NuiA complex (2, 18, 19). The three-dimensional structure of NuiA has recently been determined (20).

NucA is a monomeric enzyme with secondary structure composition, as judged by circular dichroism spectroscopy, and a metal ion cofactor requirement similar to the Serratia nuclease (2). It prefers Mn²⁺ and Co²⁺ over Mg²⁺ and shows little activity with Ni²⁺ and no activity with Zn²⁺. It has a pH optimum of 5.5-7.5 and a temperature optimum around 35 °C. With 8.4 x 10⁶ Kunitz units/mg of protein (k_cat = 2,055 s^-1), NucA is one of the most active nucleases known. For comparison, DNase I has 7.2 x 10⁵ Kunitz units/mg of protein (k_cat = 200 s^-1) (21). The general sequence preferences of NucA, e.g. avoiding d(A)·d(T) tracts (2), are similar to other nonspecific nucleases, such as the Serratia nuclease (22, 23).

NucA and Serratia nuclease share 22% sequence identity (Fig. 1), which is most pronounced in the active site region and the central multistranded -sheet core of the nuclease. According to a detailed mutational analysis, most amino acid residues important for phosphodiester bond hydrolysis by Serratia nuclease (9, 19, 24) have counterparts in NucA, suggesting that Serratia nuclease and NucA share a common mechanism of action, with differences in detail (19). It has recently become clear that the Me finger motif (in this terminology "Me" refers to the bound metal ion) is shared by a broad class of nucleases (25) which includes the highly specific homing endonuclease I-PpoI (26-28), a member of the His-Cys box family (29), and the Vibrio nuclease Vvn from Vibrio vulnificus (30, 31). Despite the overall similarities, the sequences suggest a number of significant differences, the reality of which can only be resolved by structural comparisons. For example, sequence alignment of NucA with the Serratia nuclease indicates that Arg⁵⁷ of Serratia, a residue thought to be important for stabilizing the transition state, is not present in NucA (19). Thus, NucA either uses a different mechanism for stabilizing the transition state, or there is a structural alignment that is not immediately apparent from the sequence alignment. The resolution of this and related questions can only be resolved by a complete structural determination.

View larger version (22K): [in this window] [in a new window]   FIG. 1. NucA-Serratia nuclease sequence alignment. The crystal structure of the (2-24),D121A construct of NucA begins at residue Ile36. The N-terminal His-tag as well as residues Gln25 through Ser35 are disordered. The deleted residues are indicated in magenta, the active site residues are highlighted in red, and the residues involved in coordinating the metal ions at the second binding site are colored green. The residues involved in the formation of the dimer interface in Serratia nuclease are highlighted in yellow.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
Protein Construct and Site-directed Mutagenesis—Expression of nucleases in E. coli cells generally requires some type of nuclease inactivation to limit cellular toxicity. In the present NucA studies, this was achieved by the introduction of a D121A mutation, which results in a reported 0.04% activity relative to the wild type enzyme (19). The full-length NucA sequence contains a signal peptide at the N terminus, which is responsible for the protein excretion to the periplasm. For this study we have used a construct in which N-terminal residues 2-24 were replaced by an A(H)₆M His tag, and a point mutation was introduced at position 121 to produce a D121A mutant. To obtain the D121A mutant, site-directed mutagenesis was performed using QuikChange kit (Stratagene) and verified by DNA sequencing. Residue numbering is based on the complete sequence of the enzyme (Fig. 1).

Protein Expression and Purification—Recombinant NucA was produced by transforming the plasmid into E. coli BL21 star (DE3). Cells were grown to mid-log phase (A₆₀₀ = 0.6) at 37 °C in LB medium containing 50 µg/ml carbenicillin. NucA protein expression was induced with 0.4 mM isopropyl--D-thiogalactopyranoside at 37 °C for 6 h. Cells were harvested by centrifugation (at 7,000 x g), resuspended in 50 mM Tris-HCl, pH 7.5, and 150 mM NaCl, and lysed by sonication with a Branson Sonifier 200 using a microtip probe. The lysate was centrifuged at 30,000 x g for 40 min. The nuclease was extracted from the inclusion bodies (for details, see Ref. 2) in the pellets using 50 mM Tris-HCl, pH 7.5, 6 M urea, 10 mM imidazole by soaking overnight. The urea-containing solution was recentrifuged (30,000 x g, 60 min, 4 °C), and the supernatant was applied to a nickel-nitrilotriacetic acid resin (Qiagen) equilibrated with extraction buffer and eluted with 50 mM Tris-HCl, pH 7.5, 6 M urea, and 200 mM imidazole. The eluted fraction containing protein was then concentrated using a Millipore concentrator to a desired volume of 10 ml (10 mg/ml concentration) and renatured using flash dilution of 20-fold with moderate stirring at 4 °C. The renaturing buffer containing 50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 2 mM MgCl₂, and 5 mM dithiothreitol was precooled to 4 °C. The renatured enzyme was then concentrated using an Amicon concentrator and applied to the Superdex-75 gel filtration 26 x 60-cm column and equilibrated with 50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 5 mM dithiothreitol. The major peak of absorbance at 280 nm was found to be pure NucA protein as judged by SDS-PAGE. The peak fractions were concentrated using a Millipore concentrator, and the buffer was exchanged to 20 mM Tris-HCl, pH 7.5, 200 mM NaCl.

Crystallization and Data Collection—The NucA (2-24),D121A mutant was concentrated to 10 mg/ml and dialyzed in 20 mM Tris, pH 7.5, and 200 mM NaCl. Crystals of this protein were obtained using the hanging drop vapor diffusion technique at 4 °C by mixing 2 µl of the protein solution with 2 µl of the reservoir solution consisting of 100 mM MES, pH 6.5, 10 mM ZnSO₄, 300 mM NaCl, and 35% polyethylene glycol 550. For the data set without manganese ion present, crystals were transferred and soaked in 100 mM MES, pH 6.5, 300 mM NaCl, and 35% polyethylene glycol 550. For the data set with manganese, crystals were transferred and soaked in 100 mM MES, pH 6.5, 10 mM MnCl₂, 300 mM NaCl, 35% polyethylene glycol 550, and 10 mM of the dinucleotide 5'-CG-3'.

For data collection, crystals were flash cooled by submersion in liquid nitrogen and placed on the goniometer in a stream of nitrogen gas cooled to -180 °C. Data were collected using a RU-H3R generator equipped with Osmic mirrors and a Raxis IV area detector. NucA crystallized in the orthorhombic space group P2₁2₁2₁ with one molecule/asymmetric unit.

Structure Determination and Refinement—A model for molecular replacement was created from the crystal structure of Serratia endonuclease (PDB code 1G8T [PDB] (14)) by replacing nonidentical side chains with alanines. The program Molrep (32) from CCP4 (33) was used to solve the molecular replacement problem. The model was then refined by iterative cycles of model building using the program O (34) and refinement using the program CNS (35). The quality of the final structures was assessed using the programs Procheck (36) and MolProbity (37). The statistics for the data collection and results from refinement are reported in Table I. The structure of the Mn²⁺ enzyme has been submitted to the Protein Data Bank (PDB code 1ZM8 [PDB] ).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

Structure of the Active Site in the Manganese-substituted Enzyme—To obtain a structure of NucA with Mn²⁺ in the active site, crystals grown in the presence of Zn²⁺ were transferred into solution containing no Zn²⁺ and only Mn²⁺ as a divalent cation. The bond lengths and B-factors obtained from the refinement are consistent with the identification of this ion as Mn²⁺, and we have modeled it as such. The active site revealed an octahedral coordination of the divalent ion, with an inner coordination sphere that includes the side chain carbonyl oxygen of Asn¹⁵⁵, four water molecules, and an apparent sulfate ion. The Mn-O bond distances range from 2.04 Å for the bond to Asn¹⁵⁵, to 2.36 for one of the bonds to water, with an average length of 2.19 Å (Table II). The mode of interaction with the divalent cation observed in NucA is typical of a series of other nucleases and ATPases with active sites that are optimized for binding a hydrated ion, e.g. Serratia nuclease, I-PpoI, and MutL (13, 38). In all of these examples, the active sites are optimized to interact with a hydrated metal ion rather than making direct contact with the metal ion, so that most of the protein interactions with the active site divalent ion are mediated by water molecules. For NucA, water molecule 1 (w1) is positioned by its interaction with His¹²⁴, water molecules w2 and w3 are positioned by Glu¹⁶³ (Fig. 2b), and water molecule w4 by an interaction with Gln¹⁵⁰. These positioning residues are themselves held in place by an additional network of hydrogen bonds, e.g. the second carboxyl oxygen of Glu¹⁶³ is hydrogen-bonded to the N of Trp¹⁵⁹. The only direct bond formed between the divalent ion and the protein involves the Asn¹⁵⁵ side chain carbonyl oxygen atom. In the native enzyme, the position of the Asn¹⁵⁵ side chain would probably be stabilized by the interaction with Asp¹²¹, analogous to the interaction between Asn¹¹⁹ and Asp⁸⁶ in the Serratia nuclease. However, the Asp¹²¹ side chain is not present in the D121A mutant that was studied. Apparently, the interaction between the Asn¹⁵⁵ side chain and the Mn²⁺ is sufficient to define the position of this side chain in the absence of the Asp¹²¹ interaction. The sulfate ion observed in the active site interacts with the Mn²⁺, with the side chain amide of Asn¹⁵⁵, and with the guanidino group of Arg⁹³. If the sulfate is considered as a representative for a nucleotide phosphate group, this structure implies that the Asn¹⁵⁵ and Arg⁹³ residues would also interact directly with a phosphate oxygen of the substrate. Additionally, Arg⁹³ is hydrogen bonded to Asp⁹⁵ as a part of the additional hydrogen bond network around the active site which may exist to help stabilize the active conformation of Arg⁹³. Finally, we note that although the dinucleotide 5'-CG-3' was present in the crystallization solution, no electron density could be clearly identified for the dinucleotide.

View larger version (51K): [in this window] [in a new window]   FIG. 2. Overall fold of NucA. a, stereo view of the crystal structure of NucA (2-24),D121A mutant showing the two-stranded and six-stranded -sheets (navy) and 13 -helices (lavender) forming the framework of the molecule. The two metal binding sites bind a total of three manganese ions, shown in green, and a sulfate ion, shown in yellow. b, stereo view showing the active site residues (in khaki) and the divalent metal ion (in green) with its octahedral coordination to Asn155, a sulfate ion, and four water molecules (water molecules 1-4 named as w1-w4, respectively) illustrated by orange dotted lines. The dotted lines shown in navy represent the network of hydrogen bonds near the active site. The sulfate ion is shown in yellow.

Electrostatic Surface of NucA—The electrostatic surface of the NucA is very strongly positive, with an active site cleft that is flanked by positively charged amino acids. These residues include Lys¹⁰¹, Arg¹⁰⁹, Lys¹⁸⁸, Arg¹²², Arg⁹³, and Arg¹⁶⁷ (Fig. 4). A 180° rotated view of the electrostatic surface of NucA shows the other metal ion binding site flanked by negatively charged Asp²⁴⁶, Glu²⁴⁹, and Asp²⁵⁵ on the surface.

View larger version (43K): [in this window] [in a new window]   FIG. 3. Additional metal binding sites. This stereo view of the second and third metal ion binding sites of NucA shows two metal ions (in green) and the six coordinating ligands. The Glu136 residue from a symmetry-related molecule (shown in blue) coordinates with both metal ions.

View larger version (45K): [in this window] [in a new window]   FIG. 4. Electrostatic surface of NucA. Molecular surface representations of Mn-NucA were generated using GRASP (54). Left-hand side, electrostatic potential map showing the active site cleft and the residues near the active site; right-hand side, a 180° (along the x axis) rotated view of the surface showing the second and third metal binding surface, where red and blue represent acidic and basic potentials, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

The active site of the Me finger nucleases supplies one or two Mg²⁺ ligands as identified by crystal structure analyses: Asn¹¹⁹ in Serratia nuclease (13, 14); Asn¹⁵⁵ in NucA, His⁵⁴⁴, and His⁵⁶⁹ in ColE7 (40); His¹⁰² and His¹²⁷ in ColE9 (41); Glu⁷⁹ and Asn¹²⁷ in Vvn nuclease (31); Asp²⁶² and His³⁰⁸ in CAD/DFF40 (42); Asp⁴⁰ and Asn⁶² in T4endoVII (43); Asn¹¹⁹ in I-PpoI (38); Asp⁷⁴ and Asn⁹⁶ in I-HmuI (45). Based on structure model building and biochemical data, the Mg²⁺ ligands are Asn¹⁷⁴ in EndoG (39) and Asp¹⁴⁸ and Gln¹⁷⁵ in KpnI (44). The different modes of metal ion cofactor binding allow dividing the Me finger nucleases into subgroups: Serratia nuclease, NucA, I-PpoI, and, based on computations, EndoG with an Asn residue as the sole Mg²⁺ ligand; Vvn nuclease, T4endoVII and I-HmuI with a carboxylate and a carboxamide as Mg²⁺ ligands; the colicins with two His residues as Mg²⁺ ligands and; CAD/DFF40 with a carboxylate and a histidine as Mg²⁺ ligands takes up a position between the last two groups. In all cases, direct coordination of the metal ion by protein residues is incomplete, so that the active site is optimized for binding a hydrated metal ion.

The closest known structural homolog of NucA among the Me finger nucleases and nonspecific nuclease family containing the DRGH recognition motif is the nonspecific nuclease from S. marcescens. Although Serratia nuclease and NucA have only 22% sequence identity (Fig. 1), it is the closest structure identified by the DALI server (46). The overall folds of the two enzymes are very similar (Fig. 5a). Despite this structural homology of Serratia nuclease with NucA, there are several significant differences. In particular, the Serratia nuclease, along with most other nucleases in this family, has a dimeric structure, whereas NucA is monomeric (19). The dimer interface in the Serratia nuclease includes a short loop from Arg¹³⁶ to Ile¹³⁹, a short loop and -strand region from Asn¹⁷⁸ to Tyr¹⁸⁵, and a longer stretch of residues at the C terminus of the protein (residues 225-245). As can be seen from the alignment in Fig. 1, this region of the protein is generally not well conserved and includes many gaps. His¹⁸⁴, which forms a hydrogen bond with the Ser²²⁹ and with the Pro¹⁸⁰ carbonyl in the interchain region, corresponds to Arg²²² in NucA. It has previously been noted that the H184R mutant of the Serratia nuclease is monomeric (11). The interstrand salt bridge (Asp²²⁵-Arg¹³⁶) in the Serratia structure would not exist in NucA, which has Asn and Gln residues at these positions. Similarly, the (Lys²³³-Glu²³⁹) interstrand salt bridge in Serratia would not be present in NucA. Of course, alternate stabilizing interactions could in principle be present in NucA; however, both biochemical (47) and the present crystal structure data support a monomeric structure for NucA.

View larger version (67K): [in this window] [in a new window]   FIG. 5. NucA-Serratia nuclease superposition. a, the superposition of ribbon diagrams corresponding to NucA (violet-red; this study) and one of the monomers of the Serratia nuclease (blue; chain A from 1G8T) illustrate the general similarity of the overall fold. b, stereo view of the active site residues of NucA (violet-red) and Serratia nuclease (blue), including their respective metal ions Mn2+ in NucA (peach) and Mg2+ in Serratia (gray) and the sulfate: NucA, yellow; Serratia nuclease, green. c, expanded view of the N-terminal region of NucA (residues 36-70) shown in violet-red and Serratia nuclease (residues 5-33) shown in blue. The Serratia nuclease contains a loop stabilized by a disulfide bond, whereas the NucA structure contains an -helix at a similar position.

One region of considerable sequence disparity between the two nucleases is found at the N terminus (residues 5-33 for NucA; residues 36-70 for the Serratia nuclease) (Fig. 5c). In Serratia, this region includes two cysteine residues that form a disulfide bond that has been proposed to regulate the activity of the nuclease (12). Despite the complete lack of sequence homology and the missing disulfide bond, both structures occupy a similar region of the protein, filling a generally hydrophobic pocket located on the face opposite the active site. NucA contains an -helix in place of the disulfide in Serratia nuclease. The extra secondary structure in this region of NucA may help stabilize the active conformation, whereas the presence of the disulfide in this region leads to active Serratia nuclease. The structural importance of this region in NucA was supported by our observation that a 59 construct of NucA failed to yield protein that could be refolded.

Several of the metal ion ligands present in the second and third metal ion binding site of NucA are also present in the Serratia nuclease; however, as indicated in the alignment of Fig. 1, Asp²⁵⁵ and Glu²⁶⁹ are not conserved in the Serratia sequence. No other metal binding sites were observed in two reported crystal structures of Serratia nuclease (13, 14). A survey of other related (DRGH motif-containing) nucleases also revealed no significant conservation of the residues involved in the second and third metal binding site except for Glu²⁴⁹, indicating that it is unique to NucA. Although the location of the second and third metal ion binding sites at a crystallographic interface introduces some uncertainty regarding the physiological significance of these sites, the presence of four anionic residues contributed by a single NucA molecule makes for interesting speculation. Potentially, this site could play a structural or regulatory role, it could be involved in anchoring or binding NucA to some other cellular component, similar to its role in creating a crystal contact, or it could possess an additional catalytic activity. The availability of the two metal ions for an intermolecular interaction would be consistent with the ability to interact with other substrates, suggesting that it might play a functional role.

Mechanism of Phosphodiester Bond Hydrolysis by NucA—In the present study we report the first crystal structure of NucA. This structure substantiates previous suggestions that NucA is a member of the Me family of nucleases (19), providing insight into its catalytic mechanism. Drawing analogies to the proposed mechanisms for other Me nucleases I-PpoI (38), ColE9 (41), and I-HmuI (45), the hydrolytic reaction pathway would proceed through an in-line displacement reaction mechanism whereby His¹²⁴, acting as a general base, would deprotonate a water molecule which would attack the scissile phosphate coordinated to the divalent metal ion through a nonbridging oxygen and the leaving group 3'-oxygen (Fig. 6). In both the Serratia nuclease (13, 14) and NucA structures, the proposed general base histidine forms a hydrogen bond with an inner coordination sphere water of the metal ion. Superpositions with I-PpoI (38) and I-HmuI (45), which were both crystallized in the presence of DNA, suggest that this water occupies the position of the nonbridging phosphate oxygen chelated to the metal ion. Thus, it is possible that upon substrate binding this water is replaced by the nonbridging oxygen of the phosphate, and the His¹²⁴ side chain undergoes a slight conformational change to its catalytically active conformation, similar to the position of the catalytic histidine residue in the I-PpoI structure (Fig. 6). The divalent metal ion, along with the Arg⁹³ guanidino group, would help stabilize negative charge buildup in the transition state through interactions with the nonbridging oxygen as well as enhancing the leaving group characteristics of the 3'-bridging oxygen.

View larger version (18K): [in this window] [in a new window]   FIG. 6. Catalytic mechanism. The proposed mechanism of catalysis of NucA showing the active site coordination as obtained from the present crystal structure (a) and showing a bound DNA molecule (in black) and the line of nucleophilic attack from the water molecule bound to the His124 and the stabilization of the transition state by the nearby Arg93 (b). The presumed role of Asp121 in positioning Asn155 is indicated, although this residue was mutated to alanine in the construct for which the structure was determined.

In several nucleases for which nuclease-DNA co-crystal structures are available (ColE7, His⁵⁴⁵ (40); ColE9, His¹⁰³ (41); Vvn nuclease, His⁸⁰ (31); I-PpoI, His⁹⁸ (38); and I-HmuI, His⁷⁵ (45)) a histidine residue is ideally positioned to act as a general base to activate a water molecule for the nucleophilic attack on the phosphorus atom. Most probably, this is the case in other nucleases of Me finger nuclease family. This assumption is supported by results of mutational analyses (Serratia nuclease, His⁸⁹ (24); NucA, His¹²⁴ (19); EndoG, His¹⁴³ (39); ColE7, His¹⁰³ (48); T4endoVII, His⁴¹ (49); KpnI, His¹⁴⁹ (44)) and the analysis of the pH dependence of phosphodiester bond cleavage (pH_{half-maximal activity} 6 for Serratia nuclease (24) and NucA (2)). In general, most of these nucleases do not require an additional activation or orientation of the histidine residue involved in the catalysis. It is of particular interest that, in contrast with the other active site residues, His¹²⁴ is not more rigidly positioned, e.g. by a hydrogen bond to the N of the ring. In general, a more rigid position would enhance its ability to interact with water w1, which is in the inner Mn²⁺ coordination sphere. The absence of such an interaction supports the idea of some positional variability, as illustrated, for example, in Fig. 6 as part of its catalytic function.

The position of Arg⁹³ in NucA and its interaction with the sulfate ligand, as well as the simulated DNA complex based on the Vvn structure (Fig. 7), suggest that it may play a role in positioning the substrate and in stabilizing the transition state structure. A comparison of the active sites of some Me finger nucleases suggests that in many nucleases a basic amino acid residue also is involved in neutralizing the extra negative charge on the phosphate oxygen in the transition state. Replacing Arg⁵⁷ in Serratia nuclease (24), Arg⁵ in ColE9 (50), and Arg⁶¹ in I-PpoI (51) by alanine demonstrates that these arginine residues are essential for nuclease activity (in the case of I-PpoI a large effect is only seen at pH 10). In T4endoVII three histidine residues are candidates for transition state stabilization: their substitution by other amino acid residues leads to an inactive or almost inactive enzyme (43, 52, 53). A central role for Arg⁹³ in the catalytic mechanism of NucA is supported by the observation that the NucA R93A variant has only 0.12% residual activity (19) (for comparison the Serratia nuclease R57A variant has 0.6% residual activity (24)). We therefore conclude that in the NucA-catalyzed reaction, Arg⁹³ (Fig. 6) is involved in transition state stabilization.

View larger version (29K): [in this window] [in a new window]   FIG. 7. Modeled DNA-NucA interaction. Stereo view of the modeled interaction of a duplex DNA octamer with selected residues from NucA is shown. The model is based on the alignment of the NucA structure (Arg122-Ile125) residues with Vvn (Trp78-Val81) and NucA (Met147-Arg156) with Vvn (Leu119-Gly128) residues in the Vvn·DNA structure (1OUP). The coordination of the active site Mn2+ (green) to four water molecules as well as to Asn155 and to a phosphate oxygen of the cleaved DNA strand (shown in pink) is indicated. The active site residues are shown in gray, and the complementary strand of the DNA is shown in blue.

	CONCLUSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

	FOOTNOTES

 
The atomic coordinates and structure factors (code 1ZM8) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Laboratory of Structural Biology, NIEHS, MR-01, 111 Alexnder Dr., Box 12233, Research Triangle Park, NC. Tel.: 919-541-4879; Fax: 919-541-5707; E-mail: london{at}niehs.nih.gov.

¹ The abbreviations used are: NucA, nuclease A; Me, bound metal ion; MES, 4-morpholineethanesulfonic acid; Mn2 and Mn3, metal ion 2 and 3, respectively.

	ACKNOWLEDGMENTS

 
We gratefully acknowledge the assistance of Dr. Tom Kirby in the preparation of the NucA mutant, Dr. Robert Petrovich of the Protein Expression Core Facility for protein expression, and Dr. Joseph Krahn for contributions to the structural refinement.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 

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