Bridging Water Molecules Research Articles

Sodium ion association via bridging water molecules for different charged p-phosphonated calix[4]arene bilayers

Cone conformation p-phosphonatocalix[4]arene forms bilayer and columnar arrays with sodium ions clustering above the calixarene cavity involving bridging water molecules, and a molecular ‘glue’ of either DMF or carbonate ion. The DMF has a methyl group directed into the cavity of the calixarene which takes on a 5-charge, whereas in the carbonate-containing structure the calixarene takes on an 8-charge, with a hydroxyl group coordinated to sodium directed into the cavity. The nature of the interactions in both structures has been probed using Hirshfeld surface analysis.

Unique cadmium(II) dipicolinate coordination tetramers with different Cd(II) coordination environments and number of halide ions (Cl, Br)

Cadmium(II) dipicolinate (dipic) coordination tetramers, namely [Cd2Cl(dipic)(dipicH)(H2O)4]2·2H2O (1), [Cd2Br(dipic)(dipicH)(H2O)4]2·2H2O (2), [Cd2Cl2(dipicH)2(H2O)3]2·2H2O (3), [Cd2Br2(dipicH)2(H2O)3]2·2H2O (4) were prepared. The molecular and crystal structures of 1–4 were determined by single crystal X-ray structural analysis. Tetramers 3 and 4 were also characterised by powder X-ray diffraction and thermal methods (TG, DSC and hot-stage microscopy). Tetramers 1–4 were prepared at 60°C under autogenous pressure by reaction of cadmium(II) salt (chloride or bromide) and dipicolinic acid in aqueous solution. The concentration of both reactants and the time of heating crucially effect the preparation of a particular tetramer. The tetramers 3 and 4 can be reproducibly prepared and isolated in high yield as pure compounds, whilst 1 and 2 could not be obtained in a pure form and always as the minor component in the mixture. Tetramers 1 and 2 are isostructural, containing two crystallographically independent 7-coordinated Cd(II) ions bridged by two dipicolinate ligands into a dinuclear unit. Two dinuclear units are centrosymmetrically connected via bridging water molecules into a tetrameric structure. The two 7-coordinated Cd(II) ions have different coordination environments, one having a halide ion substituted with a water molecule. Tetramers 3 and 4 are also isostructural and very similar to 1 and 2, containing 7-coordinated Cd(II) ions coordinated in the same way (including halide ion) as in 1 and 2. The difference in coordination environment of Cd(II) ions for 1 and 2, when compared to 3 and 4, is also reflected in different hydrogen bond motifs found in the crystal structures, i.e. R44(8) for 1 and 2 or R33(10) for 3 and 4.

X-Ray, Crystal Structure and Solution Phase Studies of a Polymeric SrII Compound

In the crystal structure of the title polymeric compound, (C 42 H38 N6O33 Sr 5.2(H2O)) n, five independent metal atoms (Sr1-Sr5) have different coordination environments. The Sr1 and Sr5 atoms are nine coordinated and feature distorted tricapped trigonal-prismatic and capped square-antiprismatic geometries, respectively. The rest Sr II atoms have eight coordination numbers. These units are connected via the carboxylate O atoms of mono- and di anionic forms of pyridine- 2,6-dicarboxylic acid, (pydcH 2), and bridging water molecules that resulted in the formation of polymeric layers in 3-D. In the crystal structure, non-covalent interactions consisting of hydrogen bonds (O—H···O and C—H···O), �-� (interplanar distances of 3.413(2); 3.601(2) A) and CO···� (O···� distances of 3.249(3); 3.275(3) A) stacking interactions play an important roles in stabilizing the structure. The protonation constants of propane-1, 2-diamine (1, 2-pn) and pyridine-2,6-dicarboxylate ion (pydc) 2- , the equilibrium constants for the pydc-1,2-pn proton transfer system and the stoichiometry and stability of this system with Sr 2+ ion in aqueous solution were investigated by potentiometric pH titrations. The stoichiometry of one of the most abundant complexed species in solution was found to be the same as that of the crystalline Sr II complex.

How Fast Do Microhydrated Al Clusters React: A Theoretical Study

The dynamics of the first step of the reaction of small Alm clusters m = {3,13} with up to four water molecules has been studied. The first stage in the reaction, which may ultimately result in the production of H2, is the generation of a HAlmOH·(H2O)n−1 species, which can take place through transition states of the relay type, in which the water molecules may play different catalytic roles; namely, they can be involved directly in a Grotthuss-like mechanism by forming a relay bridge, or they may lower the saddle point energy by interacting with the molecules of the relay bridge. Rate coefficients have been computed with the inclusion of tunneling effects. The results indicate that the optimal configuration (the one giving the highest values of the rate coefficient for 50 K < T < 500 K) in the case of the Al3·(H2O)n system involves three water molecules in the relay bridge. In the case of the Al13 cluster, the two-water system is the most efficient. It is remarkable that Al3 delivers much higher rates tha...

Poly[tetra-μ-aqua-diaquatetrakis[μ-(E)-2-nitrocinnamato]tetrarubidium

In the structure of the title compound, [Rb4(C9H6NO4)4(H2O)6]n, the asymmetric unit comprises four rubidium cations, two of which have an RbO7 coordination polyhedron with a monocapped distorted octa­hedral stereochemistry and two of which have a distorted RbO6 octa­hedral coordination. The bonding about both the seven-coordinate cations is similar, comprising one monodentate water mol­ecule together with three bridging water mol­ecules and three carboxyl­ate O-atom donors, two of which are bridging. The environments around the six-coordinate cations are also similar, comprising a monodentate nitro O-atom donor, a bridging water mol­ecule and four bridging carboxyl­ate O-atom donors [overall Rb—O range = 2.849 (2)–3.190 (2) Å]. The coordination leads to a two-dimensional polymeric structure extending parallel to (001), which is stabilized by inter­layer water O—H⋯O hydrogen-bonding associations to water, carboxyl and nitro O-atom acceptors, together with weak inter-ring π–π inter­actions [minimum ring centroid–centroid separation = 3.5319 (19) Å].

The structure and thermal behaviour of sodium and potassium multinuclear compounds with hexamethylenetetramine

Sodium and potassium thiocyanate complex compounds of formulae [Na(hmta)(H2O)4] 2+2 ·2SCN− (1) and [K2(hmta)(SCN)2] n (2) have been synthesized and characterised by IR spectroscopy, thermogravimetry coupled with differential thermal analysis, elemental analysis and X-ray crystallography. Each sodium and potassium cation is six co-ordinated, the sodium by one monofunctional hmta molecule, three terminal water molecules and two bridging water molecules, and the potassium by two bridging tetrafunctional hmta molecules and four bridging tetrafunctional thiocyanate ions. The coordination polyhedra of the central atoms can be described as distorted tetragonal bipyramids. The complex cations and anions of (1) are interconnected by multiple intramolecular O(water)—H···N(hmta/NCS) and O(water)—H···S hydrogen bonds to the three dimensional net. In each complex cation the intramolecular O–H···O hydrogen bonds link two terminal water molecules bonded to two metal cations. The compound (2) forms the three dimensional hybrid network in which the classical two-dimensional coordination polymers are linked by inorganic SCN− spacers to the third-dimension. Thermal analyses show that the compounds decompose gradually in three (for 1) and two (for 2) steps with formation of Na2SO4 and K2S as the final products, respectively, for 1 and 2.

Structure of (C17H22FN3O3)[MCl4]·H2O (M = Cd, Hg)

Two isostructural compounds (C17H22FN3O3)[MCl4]·H2O (M = Cd, Hg), C17H20FN3O3 is 1-ethyl-N-methyl-6-fluoride-1,4-dihydro-4-oxo-7-(4-methyl-1-piperazinyl)-3-quinoline carboxylic acid (PefH, pefloxacin), are synthesized and their crystal structure is determined. The crystals contain PefH32+ and MCl42− ions. The neighboring [MCl4]2− ions are bonded together by two bridging water molecules through four Cl...(H-O-H)2...Cl hydrogen bonds with the formation of the supramolecular motif R24(8). The supramolecular architecture of crystals is analyzed.

Structure and Excited-State Proton Transfer in the GFP S205A Mutant

To further explore excited state proton transfer (ESPT) pathways within green fluorescent protein (GFP), mutagenesis, X-ray crystallography, and time-resolved and steady-state optical spectroscopy were employed to create and study the GFP mutant S205A. In wild type GFP (wt-GFP), the proton transfer pathway includes the hydroxyl group of the chromophore, a water molecule, Ser205, and Glu222. We found that the ESPT rate constant of S205A is smaller by a factor of 20 than that of wt-GFP and larger by a factor of 2 in comparison to the ESPT rate of S205V mutant which we previously characterized. (1) High resolution crystal structures reveal that in both S205A and S205V mutants, an alternative proton transfer pathway is formed that involves the chromophore hydroxyl, a bridging water molecule, Thr203 and Glu222. The slow PT rate is explained by the long (∼3.2 Å and presumably weak) hydrogen bond between Thr203 and the water molecule, compared to the 2.7 Å normal hydrogen bond between the water molecule and Ser205 in wt-GFP. For data analysis of the experimental data from both GFP mutants, we used a two-rotamer kinetic model, assuming only one rotamer is capable of ESPT. Data analysis supports an agreement with the underlying assumption of this model.

Theoretical study of formation of ion pairs in (NH3·HCl)(H2O)6 and (NH3·HF)(H2O)6

We have performed theoretical studies on sixteen molecular cubes for both (NH3·HCl)(H2O)6 and (NH3·HF)(H2O)6. We use an empirical gauge, based upon the N–H and H–X bond lengths, to categorize the degree to which the cubes are neutral adduct or ion pair in character. On this basis, we describe all sixteen cubes of the former as highly ionized, but only five of the latter as greater than 85% ionic in character. Addition of one or two bridging water molecules to form (NH3·HF)(H2O)7 or (NH3·HF)(H2O)8 raises the percent ionic character to greater than 85% for these systems. The relative energy of the cubes can be categorized based on simple chemical principles. The computed vibrational frequency corresponding to the proton stretch in the N–H–F framework shows the highest degree of redshifting for systems near 50% ion-pair character. Molecular cubes close to neutral adduct or to ion-pair character show less redshifting of this vibrational motion.

Synthesis, structure and characterizations of a novel 3D coordination polymer constructed from rare castellated inorganic chains

Self-assembly of CdCl 2 and 1,2,4-triazole under hydrothermal condition yields a novel three-dimensional coordination polymer, namely {[Cd 8 Cl 4 (Trz) 12 (H 2 O)]·2H 2 O} n ( 1 ) (Trz = 1,2,4-triazole). Single-crystal X-ray diffraction reveals that four of the five independent Cd centers are linked by two μ 2 -Cl and two μ 3 -Cl atoms to form novel heptanuclear [Cd 7 Cl 4 ] clusters, which are connected by the bridging water molecules to generate an unprecedented 1D castellated inorganic chain. Furthermore, the fifth unique Cd center and the castellated Cd–Cl–O chain are joint to each other via six different μ 3 -Trz ligands to give a 3D organic–inorganic hybrid framework of 1 . A novel 3D hybrid coordination polymer was obtained via the self-assembly of CdCl 2 and 1,2,4-triazole under hydrothermal condition, which is constructed from unprecedented 1D castellated Cd–Cl–O inorganic chains and μ 3 -triazole ligands. ► 1D castellated inorganic chain based on heptanuclear [Cd 7 Cl 4 ] clusters. ► 3D organic–inorganic hybrid framework with rod-packing topology. ► Strong blue photoluminescence attributed to LMCT.

Water mediated self assembly of 5-(2-benzoimidazole-1-yl-ethoxy)-3-methyl-1-phenyl-1H-pyrazole-4-carboxylic acid methyl ester through CH⋯O and OH⋯N interactions

Single crystal X-ray structure analysis of 5-(2-benzoimidazole-1-yl-ethoxy)-3-methyl-1-phenyl-1H-pyrazole-4-carboxylic acid methyl ester (2).0.5H2O provided experimental proof for H⋯OC, CH⋯OH and HO⋯N interactions. The embedded water molecule bridges between molecules 2 via non-covalent interactions. Thus this molecule behaves as a preorganized host molecule for water, presenting a minimum ring-size molecular environment for water binding. Single crystal X-ray structure analysis of 5-(2-benzoimidazole-1-yl-ethoxy)-3-methyl-1-phenyl-1H-pyrazole-4-carboxylic acid methyl esterhemihydrate provided experimental proof for six hydrogen bonds by one molecule of water. The embedded water molecule bridges six molecules by two types of hydrogen bonding. Theoretical calculations showed that the conformation of the bicyclic hetero-ring alters only slightly due to the presence of the water molecule. Thus this organic molecule behaves as a very interesting preorganized host molecule for water, presenting maximum binding environment for water binding.

Do Keggin anions repulse each other in solution? The effect of solvent, counterions and ion representation investigated by free energy (PMF) simulations

Abstract To investigate whether polyoxometallate α-PW 12 O 40 3− Keggin anions (noted PW 3− ) repulse each other in water, we calculated the changes in free energy ΔG( d ) as a function of the P…P distance d (potential of mean force “PMF” calculations). As the anions approach each other, the free energy profiles are found to be quite flat, with a tiny minimum at ca. 11 A, showing that the anions can form “contact ion pairs” in the presence of either H 3 O + , UO 2 2+ or Eu 3+ counterions. The results obtained with different methodological variants (water models, PW 3− charge models, sampling procedures) support our previous finding that PW 3− ions can form dimers or oligomers in water (A. Chaumont and G. Wipff (2008) [13] ). The importance of stabilizing bridging water molecules and solute granularity is demonstrated by comparing PW 3− to S 3− spherical analogues and to PW 3+ cations (with all atomic charges of PW 3− inverted). With these analogues, a somewhat repulsive behavior (ca +2 to 3 kcal/mol) is observed at short distances. The role of water is further demonstrated by comparing PMFs in water and in methanol solution where there is no contact ion pair, but a free energy minimum at ca. 17 A, corresponding to an ion separated pair PW 3− …Eu(MeOH) 9 3+ …PW 3− . These findings are important for understanding processes like condensation and assembling of POMs and macro-ions in water or at aqueous interfaces.

Molecular Dynamics Simulations of Water Molecule-Bridges in Polar Domains of Humic Acids

The stabilizing effect of water molecule bridges on polar regions in humic substances (HSs) has been investigated by means of molecular dynamics (MD) simulations. The purpose of these investigations was to show the effect of water molecular bridges (WAMB) for cross-linking distant locations of hydrophilic groups. For this purpose, a tetramer of undecanoid fatty acids connected to a network of water molecules has been constructed, which serve as a model for spatially fixed aliphatic chains in HSs terminated by a polar (carboxyl) group. The effect of environmental polarity has been investigated by using solvents of low and medium polarity in force-field MD. A nonpolar environment simulated by n-hexane was chosen to mimic the stability of WAMB in a hydrophilic hotspot surrounded by a nonpolar environment, while the more polar acetonitrile environment was chosen to simulate a more even distribution of polarity around the carboxylic groups and the water molecules. The dynamics simulations show that the rigidity of the oligomer chains is significantly enhanced as soon as the water cluster is large enough to comprise all four carboxyl groups. Increasing the temperature leads to evaporization processes which destabilize the rigidity of the tetramer-water cluster. Embedding it into the nonpolar environment introduces a pronounced cage effect which significantly impedes removal of water molecules from the cluster region. On the other hand, a polar environment facilitates their diffusion from the polar region. One important consequence of these simulations is that although the local water network is the stabilizing factor for the organic matter matrix, the degree of stabilization is additionally affected by the presence of nonpolar surroundings.

Crystal Structure of the E2 Domain of Amyloid Precursor Protein-like Protein 1 in Complex with Sucrose Octasulfate

Missense mutations in the amyloid precursor protein (APP) gene can cause familial Alzheimer disease. It is thought that APP and APP-like proteins (APLPs) may play a role in adhesion and signal transduction because their ectodomains interact with components of the extracellular matrix. Heparin binding induces dimerization of APP and APLPs. To help explain how these proteins interact with heparin, we have determined the crystal structure of the E2 domain of APLP1 in complex with sucrose octasulfate (SOS). A total of three SOS molecules are bound to the E2 dimer. Two SOSs are bound inside a narrow intersubdomain groove, and the third SOS is bound near the two-fold axis of the protein. Mutational analyses show that most residues interacting with SOS also contribute to heparin binding, although in varying degrees; a deep pocket, defined by His-376, Lys-422, and Arg-429, and an interfacial site between Lys-314 and its symmetry mate are most important in the binding of the negatively charged polysaccharide. Comparison with a lower resolution APP structure shows that all key heparin binding residues are conserved and identically positioned, suggesting that APLP1 and APP may bind heparin similarly. In transfected HEK-293 cells, mutating residues responsible for heparin binding causes little change in the proteolysis of APP by the secretases. However, mutating a pair of conserved basic residues (equivalent to Arg-414 and Arg-415 of APLP1) immediately adjacent to the heparin binding site affects both the maturation and the processing of APP.

Vibrational Spectroscopy of Microhydrated Conjugate Base Anions

Conjugate-base anions are ubiquitous in aqueous solution. Understanding the hydration of these anions at the molecular level represents a long-standing goal in chemistry. A molecular-level perspective on ion hydration is also important for understanding the surface speciation and reactivity of aerosols, which are a central component of atmospheric and oceanic chemical cycles. In this Account, as a means of studying conjugate-base anions in water, we describe infrared multiple-photon dissociation spectroscopy on clusters in which the sulfate, nitrate, bicarbonate, and suberate anions are hydrated by a known number of water molecules. This spectral technique, used over the range of 550-1800 cm(-1), serves as a structural probe of these clusters. The experiments follow how the solvent network around the conjugate-base anion evolves, one water molecule at a time. We make structural assignments by comparing the experimental infrared spectra to those obtained from electronic structure calculations. Our results show how changes in anion structure, symmetry, and charge state have a profound effect on the structure of the solvent network. Conversely, they indicate how hydration can markedly affect the structure of the anion core in a microhydrated cluster. Some key results include the following. The first few water molecules bind to the anion terminal oxo groups in a bridging fashion, forming two anion-water hydrogen bonds. Each oxo group can form up to three hydrogen bonds; one structural result, for example, is the highly symmetric, fully coordinated SO(4)(2-)(H(2)O)(6) cluster, which only contains bridging water molecules. Adding more water molecules results in the formation of a solvent network comprising water-water hydrogen bonding in addition to hydrogen bonding to the anion. For the nitrate, bicarbonate, and suberate anions, fewer bridging sites are available, namely, three, two, and one (per carboxylate group), respectively. As a result, an earlier onset of water-water hydrogen bonding is observed. When there are more than three hydrating water molecules (n > 3), the formation of a particularly stable four-membered water ring is observed for hydrated nitrate and bicarbonate clusters. This ring binds in either a side-on (bicarbonate) or top-on (nitrate) fashion. In the case of bicarbonate, additional water molecules then add to this water ring rather than directly to the anion, indicating a preference for surface hydration. In contrast, doubly charged sulfate dianions are internally hydrated and characterized by the closing of the first hydration shell at n = 12. The situation is different for the (-)O(2)C(CH(2))(6)CO(2-) (suberate) dianion, which adapts to the hydration network by changing from a linear to a folded structure at n > 15. This change is driven by the formation of additional solute-solvent hydrogen bonds.

Poly[tri-μ2-aqua-(μ3-pyridine-2,4-dicarboxylato-κ4N,O2:O2:O2′)barium

In the polymeric title compound, [Ba(C7H3NO4)(H2O)3]n, the BaII ion is ten-coordinated in an NO9 environment by one N atom and three O atoms from three pyridine-2,4-dicarboxyl­ate (pydc) ligands and six water mol­ecules. The μ3-pydc ligands and the bridging water mol­ecules connect the Ba atoms into a layer parallel to (100). The crystal packing is stabilized by O—H⋯O and C—H⋯O hydrogen bonds.

Chiral Moieties-Oriented Single-Stranded Helical Assembly of Calix[4]azacrown Derivatives

Bridging of calix[4]arene derivatives with optically active subunits derived from chiral l/d-amino alcohols has resulted in two novel calix[4]azacrowns enantiomers S-C51H64NO7·CH3OH·H2O (S-1) and R-C51H64NO7·CH3OH·H2O (R-1), which were crystallized separately. Single crystal X-ray diffraction studies revealed that they each encapsulate a methanol molecule by weak interactions and that they form supramolecular enantiomeric helical arrangements along the crystallographic axis, which are constructed via C–H···O hydrogen bonds involving bridging water molecules. A similar helical arrangement was also found in the crystal structure of a previously reported calix[4]azacrown compound S-C108H146N2O16 (S-2).

Contribution of Active Site Residues to Substrate Hydrolysis by USP2: Insights into Catalysis by Ubiquitin Specific Proteases

The ubiquitin-specific protease (USP) structural class represents the largest and most diverse family of deubiquitinating enzymes (DUBs). Many USPs assume important biological roles and emerge as potential targets for therapeutic intervention. A clear understanding of USP catalytic mechanism requires a functional evaluation of the proposed key active site residues. Crystallographic data of ubiquitin aldehyde adducts of USP catalytic cores provided structural details on the catalytic triad residues, namely the conserved Cys and His, and a variable putative third residue, and inferred indirect structural roles for two other conserved residues (Asn and Asp), in stabilizing via a bridging water molecule the oxyanion of the tetrahedral intermediate (TI). We have expressed the catalytic domain of USP2 and probed by site-directed mutagenesis the role of these active site residues in the hydrolysis of peptide and isopeptide substrates, including a synthetic K48-linked diubiquitin substrate for which a label-free, mass spectrometry based assay has been developed to monitor cleavage. Hydrolysis of ubiquitin-AMC, a model substrate, was not affected by the mutations. Molecular dynamics simulations of USP2, free and complexed with the TI of a bona fide isopeptide substrate, were carried out. We found that Asn271 is structurally poised to directly stabilize the oxyanion developed in the acylation step, while being structurally supported by the adjacent absolutely conserved Asp575. Mutagenesis data functionally confirmed this structural role independent of the nature (isopeptide vs peptide) of the bond being cleaved. We also found that Asn574, structurally located as the third member of the catalytic triad, does not fulfill this role functionally. A dual supporting role is inferred from double-point mutation and structural data for the absolutely conserved residue Asp575, in oxyanion hole formation, and in maintaining the correct alignment and protonation of His557 for catalytic competency.

Role of bridging water molecules in GSK3β‐inhibitor complexes: Insights from QM/MM, MD, and molecular docking studies

The role of water molecules is increasingly gaining interest in drug design, and several studies have highlighted their paramount contributions to the specificity and the affinity of ligand binding. In this study, we employ the two-layer ONIOM-based quantum mechanics/molecular mechanics (QM/MM) calculations, molecular dynamics (MD) simulations, and molecular docking studies to investigate the effect of bridging water molecules at the GSK3β-inhibitors interfaces. The results obtained from the ONIOM geometry optimization and AIM analysis corroborated the presence of bridging water molecules that form hydrogen bonds with protein side chain of Thr138 and/or backbone of Gln185, and mediate interactions with inhibitors in the 10 selected GSK3β-inhibitor complexes. Subsequently, MD simulations carried out on a representative system of 1R0E demonstrated that the bridging water molecule is stable at the GSK3β-inhibitor interface and appears to contribute to the stability of the protein-inhibitor interactions. Furthermore, molecular docking studies of GSK3β-inhibitor complexes indicated that the inhibitors can increase binding affinities and the better docked conformation of inhibitors can be obtained by inclusion of the bridging water molecules, especially for the flexible inhibitors, in docking experiments into individual protein conformations. Our results elucidate the importance of bridging water molecules at the GSK3β-inhibitor interfaces and suggest that they might prove useful in rational drug design.

Defining a Structural and Kinetic Rationale for Paralogous Copies of Phenylacetate-CoA Ligases from the Cystic Fibrosis Pathogen Burkholderia cenocepacia J2315

The phenylacetic acid (PAA) degradation pathway is the sole aerobic route for phenylacetic acid metabolism in bacteria and facilitates degradation of environmental pollutants such as styrene and ethylbenzene. The PAA pathway also is implicated in promoting Burkholderia cenocepacia infections in cystic fibrosis patients. Intriguingly, the first enzyme in the PAA pathway is present in two copies (paaK1 and paaK2), yet each subsequent enzyme is present in only a single copy. Furthermore, sequence divergence indicates that PaaK1 and PaaK2 form a unique subgroup within the adenylate-forming enzyme (AFE) superfamily. To establish a biochemical rationale for the existence of the PaaK paralogs in B. cenocepacia, we present high resolution x-ray crystal structures of a selenomethionine derivative of PaaK1 in complex with ATP and adenylated phenylacetate intermediate complexes of PaaK1 and PaaK2 in distinct conformations. Structural analysis reveals a novel N-terminal microdomain that may serve to recruit subsequent PAA enzymes, whereas a bifunctional role is proposed for the P-loop in stabilizing the C-terminal domain in conformation 2. The potential for different kinetic profiles was suggested by a structurally divergent extension of the aryl substrate pocket in PaaK1 relative to PaaK2. Functional characterization confirmed this prediction, with PaaK1 possessing a lower K(m) for phenylacetic acid and better able to accommodate 3' and 4' substitutions on the phenyl ring. Collectively, these results offer detailed insight into the reaction mechanism of a novel subgroup of the AFE superfamily and provide a clear biochemical rationale for the presence of paralogous copies of PaaK of B. cenocepacia.