Catalytic Water Molecule Research Articles

The first crystal structures of a family 19 class IV chitinase: the enzyme from Norway spruce

Chitinases help plants defend themselves against fungal attack, and play roles in other processes, including development. The catalytic modules of most plant chitinases belong to glycoside hydrolase family 19. We report here x-ray structures of such a module from a Norway spruce enzyme, the first for any family 19 class IV chitinase. The bi-lobed structure has a wide cleft lined by conserved residues; the most interesting for catalysis are Glu113, the proton donor, and Glu122, believed to be a general base that activate a catalytic water molecule. Comparisons to class I and II enzymes show that loop deletions in the class IV proteins make the catalytic cleft shorter and wider; from modeling studies, it is predicted that only three N-acetylglucosamine-binding subsites exist in class IV. Further, the structural comparisons suggest that the family 19 enzymes become more closed on substrate binding. Attempts to solve the structure of the complete protein including the associated chitin-binding module failed, however, modeling studies based on close relatives indicate that the binding module recognizes at most three N-acetylglucosamine units. The combined results suggest that the class IV enzymes are optimized for shorter substrates than the class I and II enzymes, or alternatively, that they are better suited for action on substrates where only small regions of chitin chain are accessible. Intact spruce chitinase is shown to possess antifungal activity, which requires the binding module; removing this module had no effect on measured chitinase activity.

Structural and Functional Model for Ionic (K +/Na +) and pH Dependence of GTPase Activity and Polymerization of FtsZ, the Prokaryotic Ortholog of Tubulin

Bacterial cell division occurs through the formation of a protein ring (division ring) at the site of division, with FtsZ being its main component in most bacteria. FtsZ is the prokaryotic ortholog of eukaryotic tubulin; it shares GTPase activity properties and the ability to polymerize in vitro. To study the mechanism of action of FtsZ, we used molecular dynamics simulations of the behavior of the FtsZ dimer in the presence of GTP–Mg 2+ and monovalent cations. The presence of a K + ion at the GTP binding site allows the positioning of one water molecule that interacts with catalytic residues Asp235 and Asp238, which are also involved in the coordination sphere of K +. This arrangement might favor dimer stability and GTP hydrolysis. Contrary to this, Na + destabilizes the dimer and does not allow the positioning of the catalytic water molecule. Protonation of the GTP gamma-phosphate, simulating low pH, excludes both monovalent cations and the catalytic water molecule from the GTP binding site and stabilizes the dimer. These molecular dynamics predictions were contrasted experimentally by analyzing the GTPase and polymerization activities of purified Methanococcus jannaschii and Escherichia coli FtsZ proteins in vitro.

Crystal Structures of YkuI and Its Complex with Second Messenger Cyclic Di-GMP Suggest Catalytic Mechanism of Phosphodiester Bond Cleavage by EAL Domains

Cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger that is involved in the regulation of cell surface-associated traits and the persistence of infections. Omnipresent GGDEF and EAL domains, which occur in various combinations with regulatory domains, catalyze c-di-GMP synthesis and degradation, respectively. The crystal structure of full-length YkuI from Bacillus subtilis, composed of an EAL domain and a C-terminal PAS-like domain, has been determined in its native form and in complex with c-di-GMP and Ca(2+). The EAL domain exhibits a triose-phosphate isomerase-barrel fold with one antiparallel beta-strand. The complex with c-di-GMP-Ca(2+) defines the active site of the putative phosphodiesterase located at the C-terminal end of the beta-barrel. The EAL motif is part of the active site with Glu-33 of the motif being involved in cation coordination. The structure of the complex allows the proposal of a phosphodiesterase mechanism, in which the divalent cation and the general base Glu-209 activate a catalytic water molecule for nucleophilic in-line attack on the phosphorus. The C-terminal domain closely resembles the PAS-fold. Its pocket-like structure could accommodate a yet unknown ligand. YkuI forms a tight dimer via EAL-EAL and trans EAL-PAS-like domain association. The possible regulatory significance of the EAL-EAL interface and a mechanism for signal transduction between sensory and catalytic domains of c-di-GMP-specific phosphodiesterases are discussed.

Distributed Gaussian Valence Bond Surface Derived from Ab Initio Calculations.

The experimental and computational results for the tautomerization reaction of 2-pyridone are reviewed. G3, G4, CBS-APNO, and W1 model chemistries are used to generate state-of-the-art reaction energetics for the tautomerization reaction with and without catalytic water molecules in both the gas and aqueous phases. Reactive, electronic potential energy surface surfaces for use in molecular dynamics simulations were generated for these reactions following a recently improved empirical valence bond formulation. The form of molecular mechanics potentials needed for a satisfactory fit is also discussed.

Crystallographic evidence for noncoplanar catalytic aspartic acids in plasmepsin II resides in the Protein Data Bank

The carboxylate atoms of the two catalytic aspartic acid residues in aspartic proteases are nearly coplanar and in the uncomplexed form share an in-plane nucleophilic water molecule that is central to the mechanism of these enzymes. This note reports that while reviewing the electron-density maps derived from the deposited data for uncomplexed plasmepsin II from Plasmodium falciparum [Asojo et al. (2003), J. Mol. Biol. 327, 173-181; PDB code 1lf4], it was discovered that the aspartic acid residues in this structure should in fact be distinctly noncoplanar. The crystallographic model from the deposited coordinates has been re-refined against the 1.9 A resolution published diffraction data to an R(cryst) of 21.2% and an R(free) of 22.2%. The catalytic water molecule is present, but the plane of the carboxylate group of Asp214 is rotated by 66 degrees from its original position.

Structural and functional role of water molecules in bovine pancreatic phospholipase A2: a data-mining approach

The water molecules in 25 (21 high-resolution and four atomic resolution) crystal structures of bovine pancreatic phospholipase A(2) have been analyzed in order to identify the invariant water molecules and their possible roles. A total of 24 water molecules have been identified that are invariant in all 25 crystal structures examined. These include the catalytic water molecule, which is directly involved in the enzyme mechanism, and the conserved structural water molecule, which stabilizes the extended hydrogen-bonding network of the active site. Furthermore, many other water molecules stabilize the structure, whilst a few have been found to maintain the active-site geometry and provide coordination to the functionally important calcium ion. The invariant water molecules have been carefully examined and their possible roles in the structure and/or function are discussed. Molecular-dynamics studies of all 25 crystal structures have also been carried out and the results provide a good explanation of and support the findings obtained from the crystal structures.

Insights into RNA unwinding and ATP hydrolysis by the flavivirus NS3 protein

Together with the NS5 polymerase, the NS3 helicase has a pivotal function in flavivirus RNA replication and constitutes an important drug target. We captured the dengue virus NS3 helicase at several stages along the catalytic pathway including bound to single-stranded (ss) RNA, to an ATP analogue, to a transition-state analogue and to ATP hydrolysis products. RNA recognition appears largely sequence independent in a way remarkably similar to eukaryotic DEAD box proteins Vasa and eIF4AIII. On ssRNA binding, the NS3 enzyme switches to a catalytic-competent state imparted by an inward movement of the P-loop, interdomain closure and a change in the divalent metal coordination shell, providing a structural basis for RNA-stimulated ATP hydrolysis. These structures demonstrate for the first time large quaternary changes in the flaviviridae helicase, identify the catalytic water molecule and point to a beta-hairpin that protrudes from subdomain 2, as a critical element for dsRNA unwinding. They also suggest how NS3 could exert an effect as an RNA-anchoring device and thus participate both in flavivirus RNA replication and assembly.

A potent peptidomimetic inhibitor of botulinum neurotoxin serotype A has a very different conformation than SNAP-25 substrate.

Botulinum neurotoxin serotype A is the most lethal of all known toxins. Here, we report the crystal structure, along with SAR data, of the zinc metalloprotease domain of BoNT/A bound to a potent peptidomimetic inhibitor (K(i)=41 nM) that resembles the local sequence of the SNAP-25 substrate. Surprisingly, the inhibitor adopts a helical conformation around the cleavage site, in contrast to the extended conformation of the native substrate. The backbone of the inhibitor's P1 residue displaces the putative catalytic water molecule and concomitantly interacts with the "proton shuttle" E224. This mechanism of inhibition is aided by residue contacts in the conserved S1' pocket of the substrate binding cleft and by the induction of new hydrophobic pockets, which are not present in the apo form, especially for the P2' residue of the inhibitor. Our inhibitor is specific for BoNT/A as it does not inhibit other BoNT serotypes or thermolysin.

Expression and characterization of the biofilm‐related and carnosine‐hydrolyzing aminoacylhistidine dipeptidase from Vibrio alginolyticus

The biofilm-related and carnosine-hydrolyzing aminoacylhistidine dipeptidase (pepD) gene from Vibrio alginolyticus was cloned and sequenced. The recombinant PepD protein was produced and biochemically characterized and the putative active-site residues responsible for metal binding and catalysis were identified. The recombinant enzyme, which was identified as a homodimeric dipeptidase in solution, exhibited broad substrate specificity for Xaa-His and His-Xaa dipeptides, with the highest activity for the His-His dipeptide. Sequence and structural homologies suggest that the enzyme is a member of the metal-dependent metallopeptidase family. Indeed, the purified enzyme contains two zinc ions per monomer. Reconstitution of His.Tag-cleaved native apo-PepD with various metal ions indicated that enzymatic activity could be optimally restored when Zn2+ was replaced with other divalent metal ions, including Mn2+, Co2+, Ni2+, Cu2+ and Cd2+, and partially restored when Zn2+ was replaced with Mg2+. Structural homology modeling of PepD also revealed a 'catalytic domain' and a 'lid domain' similar to those of the Lactobacillus delbrueckii PepV protein. Mutational analysis of the putative active-site residues supported the involvement of His80, Asp119, Glu150, Asp173 and His461 in metal binding and Asp82 and Glu149 in catalysis. In addition, individual substitution of Glu149 and Glu150 with aspartic acid resulted in the partial retention of enzymatic activity, indicating a functional role for these residues on the catalysis and zinc ions, respectively. These effects may be necessary either for the activation of the catalytic water molecule or for the stabilization of the substrate-enzyme tetrahedral intermediate. Taken together, these results may facilitate the design of PepD inhibitors for application in antimicrobial treatment and antibody-directed enzyme prodrug therapy.

Catalytic Role of Calix[4]hydroquinone in Acetone−Water Proton Exchange: A Quantum Chemical Study of Proton Transfer via Keto−Enol Tautomerism

Calix[4]hydroquinone has recently attracted considerable interest since it forms stable tubular aggregates mediated solely by hydrogen bonding and pi-pi-stacking interactions. These aggregates trap specifically various small organic molecules and, in particular, catalyze the proton exchange of water with acetone. Using correlated quantum chemical methods, the mechanism of the observed proton exchange mediated by keto-enol tautomerism of acetone is investigated in detail. Starting with an investigation of keto-enol tautomerism of acetone-water clusters, it appears that four catalytic water molecules are optimal for the catalysis and that additional solvent water molecules lead to a decrease in efficiency. Analyses of the partial charges revealed a decrease of the polarization of the reactive hydrogen bonds due to the additional water molecules. As a next step, hydroquinone-acetone-water complexes were studied as models for the situation in the CHQ moieties. However, the computations revealed that the proton transfer reaction becomes less efficient when one catalytic water molecule is replaced by hydroquinone. Although concerted proton transfer via keto-enol tautomerism of acetone seems to be the predominant mechanism in supercritical water, it is no longer the rate-determining reaction mechanism for the catalyzed acetone-water proton exchange observed in tubular CHQ. Nevertheless, a key feature of the catalytic function of tubular CHQ has been identified to be the stiff hydrogen bonding network and the exclusion of additional solvent water molecules.

Crystal structure of a putative lysostaphin peptidase from Vibrio cholerae.

Peptidoglycan (PGN) constitutes the cell walls of virtually all bacteria, making it a target of the innate immune system. PGN is a polymer of alternating {Beta} (1{yields}4) linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), crossbridged by oligopeptide stems. Lysotaphin type enzymes are believed to cleave the glycl-glycine and glycyl-alanine bonds that occur in glycine-rich cross-bridges. Lysostaphins represent potential anti staphylococcal agents. Specifically, they can eradicate S.aureus nasal colonization in the rat model and are effective in treating methicillin-resistant S. aureus endophthalmitis in rabbits. These enzymes belong to the metalloendopeptidase family and possess a conserved HXH active site motif.

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.

Tyramine fragment binding to BACE-1

Fragment screening revealed that tyramine binds to the active site of the Alzheimer's disease drug target BACE-1. Hit expansion by selection of compounds from the Roche compound library identified tyramine derivatives with improved binding affinities as monitored by surface plasmon resonance. X-ray structures show that the amine of the tyramine fragment hydrogen-bonds to the catalytic water molecule. Structure-guided ligand design led to the synthesis of further low molecular weight compounds that are starting points for chemical leads.

Second step of hydrolytic dehalogenation in haloalkane dehalogenase investigated by QM/MM methods

Mechanistic studies on the hydrolytic dehalogenation catalyzed by haloalkane dehalogenases are of importance for environmental and industrial applications. Here, Car-Parrinello (CP) and ONIOM hybrid quantum-mechanical/molecular mechanics (QM/MM) are used investigate the second reaction step of the catalytic cycle, which comprises a general base-catalyzed hydrolysis of an ester intermediate (EI) to alcohol and free enzyme. We focus on the enzyme LinB from Sphingomonas paucimobilis UT26, for which the X-ray structure at atomic resolution is available. In agreement with previous proposals, our calculations suggest that a histidine residue (His272), polarized by glutamate (Glu132), acts as a base, accepting a proton from the catalytic water molecule and transferring it to an alcoholate ion. The reaction proceeds through a metastable tetrahedral intermediate, which shows an easily reversed reaction to the EI. In the formation of the products, the protonated aspartic acid (Asp108) can easily adopt conformation of the relaxed state found in the free enzyme. The overall free energy barrier of the reaction calculated by potential of the mean force integration using CP-QM/MM calculations is equal to 19.5 +/- 2 kcal . mol(-1). The lowering of the energy barrier of catalyzed reaction with respect to the water reaction is caused by strong stabilization of the reaction intermediate and transition state and their preorganization by electrostatic field of the enzyme.

Theoretical study on the mechanism of a ring-opening reaction of oxirane by the active-site aspartic dyad of HIV-1 protease

Two possible mechanisms of the irreversible inhibition of HIV-1 protease by epoxide inhibitors are investigated on an enzymatic model using ab initio (MP2) and density functional theory (DFT) methods (B3LYP, MPW1K and M05-2X). The calculations predict the inhibition as a general acid-catalyzed nucleophilic substitution reaction proceeding by a concerted SN2 mechanism with a reaction barrier of ca. 15-21 kcal mol(-1). The irreversible nature of the inhibition is characterized by a large negative reaction energy of ca. -17-(-24) kcal mol(-1). A mechanism with a direct proton transfer from an aspartic acid residue of the active site onto the epoxide ring has been shown to be preferred compared to one with the proton transfer from the acid catalyst facilitated by a bridging catalytic water molecule. Based on the geometry of the transition state, structural data important for the design of irreversible epoxide inhibitors of HIV-1 protease were defined. Here we also briefly discuss differences between the epoxide ring-opening reaction in HIV-1 protease and epoxide hydrolase, and the accuracy of the DFT method used.

Keto‐enol tautomerism in linear and cyclic β‐diketones: A DFT study in vacuo and in solution

AbstractDFT geometry optimizations have been performed at the B3LYP/6‐31G* level in the gas phase and at the IEF‐PCM/B3LYP/6‐31G* level in tetrahydrofuran (THF) and aqueous solutions using scaled radii for the diketo and ketoenol forms of acetylacetone and cyclohexanedione. To evaluate basis set effects, starting from the aforementioned minima, the 6‐311++G** optimized structures have been obtained. A number of complexes of both systems including one explicit water molecule have been considered up to the B3LYP/6‐311++G** level, for cyclohexanedione taking into account the B3LYP/6‐31G* basis set superposition errors as well. The diketo–ketoenol interconversion mechanisms have been investigated at the B3LYP/6‐31G* level in vacuo. Interestingly, the geometric constraint due to the presence of the ring facilitates the description of the reaction mechanism in cyclohexanedione. Despite the very different flexibility of the two systems that in the case of acetylacetone prevents a straightforward interconversion of the diketo to the most stable of its ketoenol forms, both reactions occur with a very high barrier (about 62–63 kcal/mol), unaffected by continuum solvents, that decreases by 2.5–3.5 kcal/mol after the inclusion of thermal corrections. The barriers are almost halved, becoming ∼31–35 kcal/mol, for the addition of a single water molecule according to various model reaction paths. Thermal corrections are limited (0.8–1.6 kcal/mol) for those adducts. The formation of a 1,1‐diol, explored in the case of acetylacetone, might facilitate the obtainment of the most stable diketo conformation, featuring the carbonyl groups in distinct orientations. Inclusion of dispersion and basis set effects via the G2MP2 protocol does not alter the relative stability of both system tautomers. In contrast, the G2MP2 interconversion barriers for the isolated systems in vacuo are close to the B3LYP ones, whereas they turn out to be somewhat higher than the free energy‐based B3LYP barriers in the presence of a catalytic water molecule. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2008

Mutational analysis of the nucleotide binding site of Escherichia coli dCTP deaminase

In Escherichia coli and Salmonella typhimurium about 80% of the dUMP used for dTMP synthesis is derived from deamination of dCTP. The dCTP deaminase produces dUTP that subsequently is hydrolyzed by dUTPase to dUMP and diphosphate. The dCTP deaminase is regulated by dTTP that inhibits the enzyme by binding to the active site and induces an inactive conformation of the trimeric enzyme. We have analyzed the role of residues previously suggested to play a role in catalysis. The mutant enzymes R115Q, S111C, S111T and E138D were all purified and analyzed for activity. Only S111T and E138D displayed detectable activity with a 30- and 140-fold reduction in k cat, respectively. Furthermore, S111T and E138D both showed altered dTTP inhibition compared to wild-type enzyme. S111T was almost insensitive to the presence of dTTP. With the E138D enzyme the dTTP dependent increase in cooperativity of dCTP saturation was absent, although the dTTP inhibition itself was still cooperative. Modeling of the active site of the S111T enzyme indicated that this enzyme is restricted in forming the inactive dTTP binding conformer due to steric hindrance by the additional methyl group in threonine. The crystal structure of E138D in complex with dUTP showed a hydrogen bonding network in the active site similar to wild-type enzyme. However, changes in the hydrogen bond lengths between the carboxylate and a catalytic water molecule as well as a slightly different orientation of the pyrimidine ring of the bound nucleotide may provide an explanation for the reduced activity.

X-ray absorption near edge structure study on Acutolysin-C, a zinc-metalloproteinase from Agkistrodon acutus venom: Insight into the acid-inactive mechanism

Acutolysin-C, a snake-venom zinc metalloproteinase, displays a distinct pH-dependent proteolytic activity, which has been tentatively assigned to a structural change of the zinc-containing catalytic center. In this work we compare X-ray absorption near-edge structure (XANES) experimental spectra at the Zn K-edge and theoretical calculations of solutions at different pH values. The experimental data show clear differences confirmed by a best fit using the MXAN procedure. The results show that, when pH decreases from pH 8.0 to pH 3.0, the zinc-coordinating catalytic water molecule moves far from the Glu143 residue that is considered to play an essential role in the proteolytic process. Data suggests that this is the possible mechanism that deactivates the metalloproteinase.

Molecular Basis of the Antimutagenic Activity of the House-Cleaning Inosine Triphosphate Pyrophosphatase RdgB from Escherichia coli

Inosine triphosphate pyrophosphatases, which are ubiquitous house-cleaning enzymes, hydrolyze noncanonical nucleoside triphosphates (inosine triphosphate (ITP) and xanthosine triphosphate (XTP)) and prevent the incorporation of hypoxanthine or xanthine into nascent DNA or RNA. Here we present the 1.5-Å-resolution crystal structure of the inosine triphosphate pyrophosphatase RdgB from Escherichia coli in a free state and in complex with a substrate (ITP+Ca2+) or a product (inosine monophosphate (IMP)). ITP binding to RdgB induced a large displacement of the α1 helix, closing the enzyme active site. This positions the conserved Lys13 close to the bridging oxygen between the α- and β-phosphates of the substrate, weakening the Pα–O bond. On the other side of the substrate, the conserved Asp69 is proposed to act as a base coordinating the catalytic water molecule. Our data provide insight into the molecular mechanisms of the substrate selectivity and catalysis of RdgB and other ITPases.

Structural Features that Govern Enzymatic Activity in Carbonic Anhydrase from a Low-Temperature Adapted Fish, Chionodraco hamatus

The carbonic anhydrase (CA) family of zinc metalloenzymes includes many known isozymes that have different subcellular distributions. The study described here focuses on identification of the structural features that define low-temperature adaptation in a Chionodraco hamatus protein, both for the reaction center, at an atomic level, and for the tertiary structure of the protein. To this aim, an x-ray absorption near-edge spectroscopy/Minuit x-ray absorption near-edge spectroscopy analysis of the reaction center was undertaken for both a structurally characterized human CAII and CA of C. hamatus. Higher structural levels were analyzed by sequence comparison and homology modeling. To establish whether the structural insights acquired in fish CAs are general, theoretical models were generated by homology modeling for three temperate-climate-adapted fish CAs. The measured structural differences between the two proteins are discussed in terms of the differences in the electrostatic potential between human CAII and CA of C. hamatus. We conclude that modulation of the interaction between the catalytic water molecule and the zinc ion could depend on the effect of the electrostatic potential distribution.