Hpy188I-DNA pre- and post-cleavage complexes--snapshots of the GIY-YIG nuclease mediated catalysis

The GIY-YIG nuclease domain is present in all kingdoms of life and has diverse functions. It is found in the eukaryotic flap endonuclease and Holliday junction resolvase Slx1-Slx4, the prokaryotic nucleotide excision repair proteins UvrC and Cho, and in proteins of 'selfish' genetic elements. Here we present the structures of the ternary pre- and post-cleavage complexes of the type II GIY-YIG restriction endonuclease Hpy188I with DNA and a surrogate or catalytic metal ion, respectively. Our structures suggest that GIY-YIG nucleases catalyze DNA hydrolysis by a single substitution reaction. They are consistent with a previous proposal that a tyrosine residue (which we expect to occur in its phenolate form) acts as a general base for the attacking water molecule. In contrast to the earlier proposal, our data identify the general base with the GIY and not the YIG tyrosine. A conserved glutamate residue (Glu149 provided in trans in Hpy188I) anchors a single metal cation in the active site. This metal ion contacts the phosphate proS oxygen atom and the leaving group 3'-oxygen atom, presumably to facilitate its departure. Taken together, our data reveal striking analogy in the absence of homology between GIY-YIG and ββα-Me nucleases.     

Behavior of BsoBI endonuclease in the presence and absence of DNA

BsoBI is a type II restriction endonuclease belonging to the EcoRI family. There is only one previously published X-ray structure for this endonuclease: it shows a homodimer of BsoBI completely encircling DNA in a tunnel. In this work, molecular dynamics simulations were employed to elucidate possible ways in which DNA is loaded into this complex prior to its cleavage. We found that the dimer does not open spontaneously when DNA is removed from the complex on the timescale of our simulations (~ 0.5 μs). A biased simulation had to be used to facilitate the opening, which revealed a possible way for the two catalytic domains to separate. The α-helices connecting the catalytic and helical domains were found to act as a hinge during the separation. In addition, we found that the opening of the BsoBI dimer was influenced by the type of counterions present in the environment. A reference simulation of the BsoBI/DNA complex further showed spontaneous reorganization of the active sites due to the binding of solvent ions, which led to an active-site structure consistent with other experimental structures of type II restriction endonucleases determined in the presence of metal ions.     

Mva1269I: a monomeric type IIS restriction endonuclease from Micrococcus varians with two EcoRI- and FokI-like catalytic domains

Type II restriction endonuclease Mva1269I recognizes an asymmetric DNA sequence 5'-GAATGCN / -3'/5'-NG / CATTC-3' and cuts top and bottom DNA strands at positions, indicated by the "/" symbol. Most restriction endonucleases require dimerization to cleave both strands of DNA. We found that Mva1269I is a monomer both in solution and upon binding of cognate DNA. Protein fold-recognition analysis revealed that Mva1269I comprises two "PD-(D/E)XK" domains. The N-terminal domain is related to the 5'-GAATTC-3'-specific restriction endonuclease EcoRI, whereas the C-terminal one resembles the nonspecific nuclease domain of restriction endonuclease FokI. Inactivation of the C-terminal catalytic site transformed Mva1269I into a very active bottom strand-nicking enzyme, whereas mutants in the N-terminal domain nicked the top strand, but only at elevated enzyme concentrations. We found that the cleavage of the bottom strand is a prerequisite for the cleavage of the top strand. We suggest that Mva1269I evolved the ability to recognize and to cleave its asymmetrical target by a fusion of an EcoRI-like domain, which incises the bottom strand within the target, and a FokI-like domain that completes the cleavage within the nonspecific region outside the target sequence. Our results have implications for the molecular evolution of restriction endonucleases, as well as for perspectives of engineering new restriction and nicking enzymes with asymmetric target sites.     

Evidence of Horizontal Transfer of the EcoO109I Restriction-Modification Gene to Escherichia coli Chromosomal DNA

A DNA fragment carrying the genes coding for EcoO109I endonuclease and EcoO109I methylase, which recognize the nucleotide sequence 5'-(A/G)GGNCC(C/T)-3', was cloned from the chromosomal DNA of Escherichia coli H709c. The EcoO109I restriction-modification (R-M) system was found to be inserted between the int and psu genes from satellite bacteriophage P4, which were lysogenized in the chromosome at the P4 phage attachment site of the corresponding leuX gene observed in E. coli K-12 chromosomal DNA. The sid gene of the prophage was inactivated by insertion of one copy of IS21. These findings may shed light on the horizontal transfer and stable maintenance of the R-M system.     

Mechanism of hydrolysis of phosphate esters by the dimetal center of 5'-nucleotidase based on crystal structures

5'-Nucleotidase belongs to a large superfamily of distantly related dinuclear metallophosphatases including the Ser/Thr protein phosphatases and purple acid phosphatases. The protein undergoes a 96 degrees domain rotation between an open (inactive) and a closed (active) enzyme form. Complex structures of the closed form with the products adenosine and phosphate, and with the substrate analogue inhibitor alpha,beta-methylene ADP, have been determined at 2.1 A and 1.85 A resolution, respectively. In addition, a complex of the open form of 5'-nucleotidase with ATP was analyzed at a resolution of 1.7 A. These structures show that the adenosine group binds to a specific binding pocket of the C-terminal domain. The adenine ring is stacked between Phe429 and Phe498. The N-terminal domain provides the ligands to the dimetal cluster and the conserved His117, which together form the catalytic core structure. However, the three C-terminal arginine residues 375, 379 and 410, which are involved in substrate binding, may also play a role in transition-state stabilization. The beta-phosphate group of the inhibitor is terminally coordinated to the site 2 metal ion. The site 1 metal ion coordinates a water molecule which is in an ideal position for a nucleophilic attack on the phosphorus atom, assuming an in-line mechanism of phosphoryl transfer. Another water molecule bridges the two metal ions.     

Catalytic features of the botulinum neurotoxin A light chain revealed by high resolution structure of an inhibitory peptide complex

The Clostridium botulinum neurotoxin serotype A light chain (BoNT/A-LC) is a Zn(II)-dependent metalloprotease that blocks the release of acetylcholine at the neuromuscular junction by cleaving SNAP-25, one of the SNARE proteins required for exocytosis. Because of the potential for use of the toxin in bioterrorism and the increasingly widespread application of the toxin in the medical field, there is significant interest in the development of small-molecule inhibitors of the metalloprotease. Efforts to design such inhibitors have not benefited from knowledge of how peptides bind to the active site since the enzyme-peptide structures available previously either were not occupied in the vicinity of the catalytic Zn(II) ion or did not represent the product of SNAP-25 substrate cleavage. Herein we report the 1.4 A-resolution X-ray crystal structure of a complex between the BoNT/A-LC and the inhibitory peptide N-Ac-CRATKML, the first structure of the light chain with an inhibitory peptide bound at the catalytic Zn(II) ion. The peptide is bound with the Cys S gamma atom coordinating the metal ion. Surprisingly, the cysteine sulfur is oxidized to the sulfenic acid form. Given the unstable nature of this species in solution, is it likely that oxidation occurs on the enzyme. In addition to the peptide-bound structure, we report two structures of the unliganded light chain with and without the Zn(II) cofactor bound at 1.25 and 1.20 A resolution, respectively. The two structures are nearly identical, confirming that the Zn(II) ion plays a purely catalytic role. Additionally, the structure of the Zn(II)-bound uncomplexed enzyme allows identification of the catalytic water molecule and a second water molecule that occupies the same position as the peptidic oxygen in the tetrahedral intermediate. This observation suggests that the enzyme active site is prearranged to stabilize the tetrahedral intermediate of the protease reaction.     

Role of the catalytic metal during polymerization by DNA polymerase lambda

The incorporation of dNMPs into DNA by polymerases involves a phosphoryl transfer reaction hypothesized to require two divalent metal ions. Here we investigate this hypothesis using as a model human DNA polymerase lambda (Pol lambda), an enzyme suggested to be activated in vivo by manganese. We report the crystal structures of four complexes of human Pol lambda. In a 1.9 A structure of Pol lambda containing a 3'-OH and the non-hydrolyzable analog dUpnpp, a non-catalytic Na+ ion occupies the site for metal A and the ribose of the primer-terminal nucleotide is found in a conformation that positions the acceptor 3'-OH out of line with the alpha-phosphate and the bridging oxygen of the pyrophosphate leaving group. Soaking this crystal in MnCl2 yielded a 2.0 A structure with Mn2+ occupying the site for metal A. In the presence of Mn2+, the conformation of the ribose is C3'-endo and the 3'-oxygen is in line with the leaving oxygen, at a distance from the phosphorus atom of the alpha-phosphate (3.69 A) consistent with and supporting a catalytic mechanism involving two divalent metal ions. Finally, soaking with MnCl2 converted a pre-catalytic Pol lambda/Na+ complex with unreacted dCTP in the active site into a product complex via catalysis in the crystal. These data provide pre- and post-transition state information and outline in a single crystal the pathway for the phosphoryl transfer reaction carried out by DNA polymerases.     

Restriction endonuclease EcoO109 from Escherichia coli H709c with heptanucleotide recognition site 5'-PuG/GNCCPy

A new restriction endonuclease, EcoO109, has been isolated from Escherichia coli H709c by polyethyleneimine (PEI) precipitation, DEAE-cellulose chromatography and heparin agarose chromatography. The yield was high, more than 3000 units/g of wet cells. The EcoO109 endonuclease recognizes and cleaves a nucleotide sequence of (formula: see text), in the presence of 10 mM Mg2+. The enzyme will be useful for structural analysis and molecular cloning of DNA because of the stability, high yield and easy handling of the producer strain.     

Metal ions bound at the active site of the junction-resolving enzyme T7 endonuclease I

T7 endonuclease I is a nuclease that is selective for the structure of the four-way DNA junction. The active site is similar to those of a number of restriction enzymes. We have solved the crystal structure of endonuclease I with a wild-type active site. Diffusion of manganese ions into the crystal revealed two peaks of electron density per active site, defining two metal ion-binding sites. Site 1 is fully occupied, and the manganese ion is coordinated by the carboxylate groups of Asp55 and Glu65, and the main chain carbonyl of Thr66. Site 2 is partially occupied, and the metal ion has a single protein ligand, the remaining carboxylate oxygen atom of Asp55. Isothermal titration calorimetry showed the sequential exothermic binding of two manganese ions in solution, with dissociation constants of 0.58 +/- 0.019 and 14 +/- 1.5 mM. These results are consistent with a two metal ion mechanism for the cleavage reaction, in which the hydrolytic water molecule is contained in the first coordination sphere of the site 1-bound metal ion.     

Characterization and overproduction of EcoO109I methyltransferase.

In order to characterize EcoO109I methyltransferase, a recombinant Escherichia coli clone that overproduces the enzyme was constructed. The coding region of M.EcoO109I was joined to the lac promoter of an expression vector, pUC118, and the resulting plasmid was introduced into E. coli HB101. M.EcoO109I was purified homogeneously from IPTG-induced cells, and was found to consist of a monomer subunit. M.EcoO109I uniquely methylates the inner deoxycytidylate residue in the sequence 5'-(A/G)GGNCC(C/T)-3' to produce 5-methylcytosine. The enzyme was most active at pH 8.0-8.5 and 50 degrees C. The enzyme activity was not affected by the addition of Mg2+ or EDTA.     

Categoric prediction of metal ion mechanisms in the active sites of 17 select type II restriction endonucleases

Recently determined crystal structures of type II restriction endonucleases have produced a plethora of information on the basis for target site sequence selectivity. The positioning and role of metal ions in DNA recognition sites might reflect important properties of protein-DNA interaction. 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. We have examined the interatomic distances between metal ions and proposed key catalytic residues in the binding sites of seventeen type II restriction endonucleases whose crystal structures are documented in literature. The summary and critical evaluation of structural assignments and predictions made earlier have been useful to group these enzymes. All the enzymes used for this study have been categorized on the basis of the number of metal ions identified in their crystal structures. Among 17 experimentally characterized (not putative) type II REases, whose apparently full-length sequences are available in REBASE, we predict 8 (47%) to follow the single metal ion mechanism, 5 to follow the two metal ion mechanism, 2, the three metal ion mechanism, 1, the four metal ion mechanism and 1 the six metal ion mechanism.     

Probing the two-metal ion mechanism in the restriction endonuclease BamHI

The choreography of restriction endonuclease catalysis is a long-standing paradigm in molecular biology. Bivalent metal ions are required almost for all PD..D/ExK type enzymes, but the number of cofactors essential for the DNA backbone scission remained ambiguous. On the basis of crystal structures and biochemical data for various restriction enzymes, three models have been developed that assign critical roles for one, two, or three metal ions during the phosphodiester hydrolysis. To resolve this apparent controversy, we investigated the mechanism of BamHI catalysis using quantum mechanical/molecular mechanical simulation techniques and determined the activation barriers of three possible pathways that involve a Glu-113 or a neighboring water molecule as a general base or an external nucleophile that penetrated from bulk solution. The extrinsic mechanism was found to be the most favorable with an activation free energy of 23.4 kcal/mol, in reasonable agreement with the experimental data. On the basis of the effect of the individual metal ions on the activation barrier, metal ion A was concluded to be pivotal for the reaction, while the enzyme lacking metal ion B still has moderate efficiency. Thus, we propose that the catalytic scheme of BamHI does not involve a general base for nucleophile generation and requires one obligatory metal ion for catalysis that stabilizes the attacking nucleophile and coordinates it throughout the nucleophilic attack. Such a model may also explain the variation in the number of metal ions in the crystal structures and thus could serve as a framework for a unified catalytic scheme of type II restriction endonucleases.     

X-ray crystallographic analyses of complexes between bovine beta-trypsin and Schiff base copper(II) or iron(III) chelates

To establish the structural basis underlying the activity of a novel series of metal-chelate trypsin inhibitors, the structures of p-amidinosalicylidene-l-alaninato(aqua)copper(II) (1a), m-amidinosalicylidene-l-alaninato(aqua)copper(II) (1b), bis(p-amidinosalicylidene-l-alaninato)iron(III) (2a), and bis(m-amidinosalicylidene-l-alaninato)iron(III) (2b) bound to bovine beta-trypsin were studied by X-ray crystallography. The amidinium group of the inhibitor donates hydrogen bonds to Asp189, Gly219 and Ser190, as seen before in trypsin-benzamidine complexes. The copper(II) ion of 1a is situated away from trypsin's catalytic triad residues, and is octahedrally coordinated by a Schiff base and three water molecules. In contrast, the copper(II) ion of 1b is situated close to the catalytic triad and adopts a square pyramidal coordination geometry. The iron(III) ion of 2a is octahedrally coordinated by two Schiff base ligands and, like the copper(II) ion of 1a, is situated away from the catalytic triad. The p-amidinophenyl ring of a second Schiff base ligand of 2a is directed toward a hydrophobic groove formed by Trp215 and Leu99. Finally, the iron(III) ion of 2b appears to be replaced by magnesium(II), which is octahedrally coordinated by a Schiff base, Gln192 and two water molecules. One of the Schiff base ligands seen in the trypsin-2a complex or in the unbound form of 2b is replaced by water molecules and Gln192. His57 and Ser195 form water-mediated interactions with the magnesium(II) ion of 2b, and Ser195 also forms a hydrogen bond with the phenolic oxygen atom of the Schiff base ligand. These structures reveal a novel mode of interaction between metal-chelate inhibitors and serine proteases, thus providing a structural basis for the development of more potent inhibitors against a variety of trypsin-like enzymes.     

Synthesis,characterization, crystal structure of the coordination polymer Zn(II) with thiosemicarbazone of glyoxalic acid and their inhibitory properties against some metabolic enzymes

A new coordination polymer Zn(II) with thiosemicarbazone glyoxalic acid H₂GAT was obtained in this study. According to the X-ray diffraction data, the coordination of the Zn(II) ion is carried out by one sulfur atom, in the thiol form, one nitrogen atom of the azomethine group and two oxygen atoms of the carboxylate groups, one of which belongs to neighbouring complex molecule. The oxygen atom of the water molecule completes Zn(II) ion environment to a distorted square-pyramidal structure. The binding of the monomer complex into polimer occurs through the bridge oxygen atom of carboxylate group. This complex is effective inhibitor of the α-glycosidase, butyrylcholinesterase (BChE), cytosolic carbonic anhydrase I and II isoforms (hCA I and II), and acetylcholinesterase enzymes (AChE) enzymes with Ki values of 1.45 ± 0.23 µM for hCA I, 2.04 ± 0.11 µM for hCA II, 3.47 ± 0.88 µM for α-glycosidase, 0.47 ± 0.10 µM for BChE, and 0.58 ± 0.13 µM for AChE, respectively.     

Genetics of cosQ, the DNA-packaging termination site of phage lambda: local suppressors and methylation effects

The cos site of the bacteriophage lambda chromosome contains the sites required for DNA processing and packaging during virion assembly. cos is composed of three subsites, cosQ, cosN, and cosB. cosQ is required for the termination of chromosome packaging. Previous studies have shown cosQ mutations to be suppressed in three ways: by a local suppressor within cosQ; by an increase in the length of the lambda chromosome; and by missense mutations affecting the prohead's portal protein, gpB. In the first study reported here, revertants of a set of cosQ mutants were screened for suppressors, and cis-acting suppressors of cosQ mutations were studied; these included second-site cosQ point mutations, base-pair insertions within cosQ, and an additional genome-lengthening suppressor. The 7-bp-long cosQ, with the sequence 5'-GGGTCCT-3', coincides exactly with the recognition site for the EcoO109I restriction/methylation system, which has the consensus sequence 5'-PuGGNCCPy-3'. In a second study, EcoO109I methylation was found to strongly interfere with the residual cosQ function of leaky cosQ mutants. cis-acting suppressors that overcome methylation-associated defects, including a methylation-dependent suppressor, were also isolated. Models of cosQ suppression are presented.     

A trimetal site and substrate distortion in a family II inorganic pyrophosphatase

We report the first crystal structures of a family II pyrophosphatase complexed with a substrate analogue, imidodiphosphate (PNP). These provide new insights into the catalytic reaction mechanism of this enzyme family. We were able to capture the substrate complex both by fluoride inhibition and by site-directed mutagenesis providing complementary snapshots of the Michaelis complex. Structures of both the fluoride-inhibited wild type and the H98Q variant of the PNP-Bacillus subtilis pyrophosphatase complex show a unique trinuclear metal center. Each metal ion coordinates a terminal oxygen on the electrophilic phosphate and a lone pair on the putative nucleophile, thus placing it in line with the scissile bond without any coordination by protein. The nucleophile moves further away from the electrophilic phosphorus site, to the opposite side of the trimetal plane, upon binding of substrate. In comparison with earlier product complexes, the side chain of Lys296 has swung in and so three positively charged side chains, His98, Lys205 and Lys296, now surround the bridging nitrogen in PNP. Finally, one of the active sites in the wild-type structure appears to show evidence of substrate distortion. Binding to the enzyme may thus strain the substrate and thus enhance the catalytic rate.     

Insight into the catalytic mechanism of DNA polymerase beta: structures of intermediate complexes

The catalytic reaction mediated by DNA polymerases is known to require two Mg(II) ions, one associated with dNTP binding and the other involved in metal ion catalysis of the chemical step. Here we report a functional intermediate structure of a DNA polymerase with only one metal ion bound, the DNA polymerase beta-DNA template-primer-chromium(III).2'-deoxythymidine 5'-beta,gamma-methylenetriphosphate [Cr(III).dTMPPCP] complex, at 2.6 A resolution. The complex is distinct from the structures of other polymerase-DNA-ddNTP complexes in that the 3'-terminus of the primer has a free hydroxyl group. Hence, this structure represents a fully functional intermediate state. Support for this contention is provided by the observation of turnover in biochemical assays of crystallized protein as well as from the determination that soaking Pol beta crystals with Mn(II) ions leads to formation of the product complex, Pol beta-DNA-Cr(III).PCP, whose structure is also reported. An important feature of both structures is that the fingers subdomain is closed, similar to structures of other ternary complexes in which both metal ion sites are occupied. These results suggest that closing of the fingers subdomain is induced specifically by binding of the metal-dNTP complex prior to binding of the catalytic Mg(2+) ion. This has led us to reevaluate our previous evidence regarding the existence of a rate-limiting conformational change in Pol beta's reaction pathway. The results of stopped-flow studies suggest that there is no detectable rate-limiting conformational change step.     

Ca2+ binding in the active site of HincII: implications for the catalytic mechanism

The 2.8 A crystal structure of the type II restriction endonuclease HincII bound to Ca(2+) and cognate DNA containing GTCGAC is presented. The DNA is uncleaved, and one calcium ion is bound per active site, in a position previously described as site I in the related blunt cutting type II restriction endonuclease EcoRV [Horton, N. C., Newberry, K. J., and Perona, J. J. (1998) Proc. Natl. Acad. Sci. U.S.A. 95 (23), 13489-13494], as well as that found in other related enzymes. Unlike the site I metal in EcoRV, but similar to that of PvuII, NgoMIV, BamHI, BglII, and BglI, the observed calcium cation is directly ligated to the pro-S(p) oxygen of the scissile phosphate. A calcium ion-ligated water molecule is well positioned to act as the nucleophile in the phosphodiester bond cleavage reaction, and is within hydrogen bonding distance of the conserved active site lysine (Lys 129), as well as the pro-R(p) oxygen of the phosphate group 3' of the scissile phosphate, suggesting possible roles for these groups in the catalytic mechanism. Kinetic data consistent with an important role for the 3'-phosphate group in DNA cleavage by HincII are presented. The previously observed sodium ion [Horton, N. C., Dorner, L. F., and Perona, J. J. (2002) Nat. Struct. Biol. 9, 42-47] persists in the active sites of the Ca(2+)-bound structure; however, kinetic data show little effect on the single-turnover rate of DNA cleavage in the absence of Na(+) ions.     

A new use for a familiar fold: the X-ray crystal structure of GTP-bound GTP cyclohydrolase III from Methanocaldococcus jannaschii reveals a two metal ion catalytic mechanism

GTP cyclohydrolase (GCH) III from Methanocaldococcus jannaschii, which catalyzes the conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (FAPy), has been shown to require Mg2+ for catalytic activity and is activated by monovalent cations such as K+ and ammonium [Graham, D. E., Xu, H., and White, R. H. (2002) Biochemistry 41, 15074-15084]. The reaction is formally identical to that catalyzed by a GCH II ortholog (SCO 6655) from Streptomyces coelicolor; however, SCO 6655, like other GCH II proteins, is a zinc-containing protein. The structure of GCH III complexed with GTP solved at 2 A resolution clearly shows that GCH III adopts a distinct fold that is closely related to the palm domains of phosphodiesterases, such as DNA polymerase I. GCH III is a tetramer of identical subunits; each monomer is composed of an N- and a C-terminal domain that adopt nearly superimposible structures, suggesting that the protein has arisen by gene duplication. Three metal ions were located in the active site, two of which occupy positions that are analogous to those occupied by divalent metal ions in the structures of a number of palm domain containing proteins, such as DNA polymerase I. Two conserved Asp residues that coordinate the metal ions, which are also found in palm domain containing proteins, are observed in GCH III. Site-directed variants (Asp-->Asn) of these residues in GCH III are less active than wild-type. The third metal ion, most likely a potassium ion, is involved in substrate recognition through coordination of O6 of GTP. The arrangement of the metal ions in the active site suggests that GCH III utilizes two metal ion catalysis. The structure of GCH III extends the repertoire of possible reactions with a palm fold to include cyclohydrolase chemistry.     

Experimental evidence for a metallohydrolase mechanism in which the nucleophile is not delivered by a metal ion: EPR spectrokinetic and structural studies of aminopeptidase from Vibrio proteolyticus

Metallohydrolases catalyse some of the most important reactions in biology and are targets for numerous chemotherapeutic agents designed to combat bacterial infectivity, antibiotic resistance, HIV infectivity, tumour growth, angiogenesis and immune disorders. Rational design of inhibitors of these enzymes with chemotherapeutic potential relies on detailed knowledge of the catalytic mechanism. The roles of the catalytic transition ions in these enzymes have long been assumed to include the activation and delivery of a nucleophilic hydroxy moiety. In the present study, catalytic intermediates in the hydrolysis of L-leucyl-L-leucyl-L-leucine by Vibrio proteolyticus aminopeptidase were characterized in spectrokinetic and structural studies. Rapid-freeze-quench EPR studies of reaction products of L-leucyl-L-leucyl-L-leucine and Co(II)-substituted aminopeptidase, and comparison of the EPR data with those from structurally characterized complexes of aminopeptidase with inhibitors, indicated the formation of a catalytically competent post-Michaelis pre-transition state intermediate with a structure analogous to that of the inhibited complex with bestatin. The X-ray crystal structure of an aminopeptidase-L-leucyl-L-leucyl-L-leucine complex was also analogous to that of the bestatin complex. In these structures, no water/hydroxy group was observed bound to the essential metal ion. However, a water/hydroxy group was clearly identified that was bound to the metal-ligating oxygen atom of Glu152. This water/hydroxy group is proposed as a candidate for the active nucleophile in a novel metallohydrolase mechanism that shares features of the catalytic mechanisms of aspartic proteases and of B2 metallo-beta-lactamases. Preliminary studies on site-directed variants are consistent with the proposal. Other features of the structure suggest roles for the dinuclear centre in geometrically and electrophilically activating the substrate.