High resolution crystal structures of human kynurenine aminotransferase-I bound to PLP cofactor, and in complex with aminooxyacetate.

In this study, we report two high-resolution structures of the pyridoxal 5' phosphate (PLP)-dependent enzyme kynurenine aminotransferase-I (KAT-I). One is the native structure with the cofactor in the PLP form bound to Lys247 with the highest resolution yet available for KAT-I at 1.28 Å resolution, and the other with the general PLP-dependent aminotransferase inhibitor, aminooxyacetate (AOAA) covalently bound to the cofactor at 1.54 Å. Only small conformational differences are observed in the vicinity of the aldimine (oxime) linkage with which the PLP forms the Schiff base with Lys247 in the 1.28 Å resolution native structure, in comparison to other native PLP-bound structures. We also report the inhibition of KAT-1 by AOAA and aminooxy-phenylpropionic acid (AOPP), with IC50s of 13.1 and 5.7 µM, respectively. The crystal structure of the enzyme in complex with the inhibitor AOAA revealed that the cofactor is the PLP form with the external aldimine linkage. The location of this oxime with the PLP, which forms in place of the native internal aldimine linkage of PLP of the native KAT-I, is away from the position of the native internal aldimine, with the free Lys247 substantially retaining the orientation of the native structure. Tyr101, at the active site, was observed in two conformations in both structures.

Insights into an evolutionary strategy leading to antibiotic resistance.

Metallo-ß-lactamases (MBLs) with activity towards a broad-spectrum of ß-lactam antibiotics have become a major threat to public health, not least due to their ability to rapidly adapt their substrate preference. In this study, the capability of the MBL AIM-1 to evade antibiotic pressure by introducing specific mutations was probed by two alternative methods, i.e. site-saturation mutagenesis (SSM) of active site residues and in vitro evolution. Both approaches demonstrated that a single mutation in AIM-1 can greatly enhance a pathogen's resistance towards broad spectrum antibiotics without significantly compromising the catalytic efficiency of the enzyme. Importantly, the evolution experiments demonstrated that relevant amino acids are not necessarily in close proximity to the catalytic centre of the enzyme. This observation is a powerful demonstration that MBLs have a diverse array of possibilities to adapt to new selection pressures, avenues that cannot easily be predicted from a crystal structure alone.

Compensatory stabilizing role of surface mutations during the directed evolution of dienelactone hydrolase for enhanced activity.

Directed evolution is a common tool employed to generate enzymes suitable for industrial use. High thermal stability is often advantageous or even a requirement for biocatalysts, as such the evolution of protein stability is of practical as well as academic interest. Even when evolving enzymes for new or improved catalytic functions, stability is an important factor since it can limit the accumulation rate and number of desired active site mutations. Dienelactone hydrolase, a small monomeric protein, has been previously evolved via a three-stage process to possess enhanced activity and specificity toward non-physiological substrates. In addition to seven active site mutations there were three surface mutations that were thought to increase the stability of the enzyme and compensate for the destabilizing active site mutations. Here, the individual influence of the three surface mutations--Q110L, Y137C and N154D--on the thermal and chemical stability of DLH has been assessed. While the Q110L and N154D mutations improved the thermal stability, the influence of the Y137C mutation was more complex. Individually it was destabilizing both thermally and chemically, but when in the presence of the Q110L and N154D mutations its effect was neutralized in relation to thermal but not chemical stability. In the context of a directed evolution experiment, these compensatory surface mutations play important roles. However, our results show that detrimental mutations can arise, thus the simultaneous monitoring of stability changes while evolving enzymes for enhanced catalytic properties can be beneficial.

Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search.

The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/ß hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.

Recommending Hartree-Fock theory with London-dispersion and basis-set-superposition corrections for the optimization or quantum refinement of protein structures.

We demonstrate the importance of properly accounting for London dispersion and basis-set-superposition error (BSSE) in quantum-chemical optimizations of protein structures, factors that are often still neglected in contemporary applications. We optimize a portion of an ensemble of conformationally flexible lysozyme structures obtained from highly accurate X-ray crystallography data that serve as a reliable benchmark. We not only analyze root-mean-square deviations from the experimental Cartesian coordinates, but also, for the first time, demonstrate how London dispersion and BSSE influence crystallographic R factors. Our conclusions parallel recent recommendations for the optimization of small gas-phase peptide structures made by some of the present authors: Hartree-Fock theory extended with Grimme's recent dispersion and BSSE corrections (HF-D3-gCP) is superior to popular density functional theory (DFT) approaches. Not only are statistical errors on average lower with HF-D3-gCP, but also the convergence behavior is much better. In particular, we show that the BP86/6-31G* approach should not be relied upon as a black-box method, despite its widespread use, as its success is based on an unpredictable cancellation of errors. Using HF-D3-gCP is technically straightforward, and we therefore encourage users of quantum-chemical methods to adopt this approach in future applications.

Crystallization of dienelactone hydrolase in two space groups: structural changes caused by crystal packing.

Dienelactone hydrolase (DLH) is a monomeric protein with a simple α/ß-hydrolase fold structure. It readily crystallizes in space group P212121 from either a phosphate or ammonium sulfate precipitation buffer. Here, the structure of DLH at 1.85 Šresolution crystallized in space group C2 with two molecules in the asymmetric unit is reported. When crystallized in space group P212121 DLH has either phosphates or sulfates bound to the protein in crucial locations, one of which is located in the active site, preventing substrate/inhibitor binding. Another is located on the surface of the enzyme coordinated by side chains from two different molecules. Crystallization in space group C2 from a sodium citrate buffer results in new crystallographic protein-protein interfaces. The protein backbone is highly similar, but new crystal contacts cause changes in side-chain orientations and in loop positioning. In regions not involved in crystal contacts, there is little change in backbone or side-chain configuration. The flexibility of surface loops and the adaptability of side chains are important factors enabling DLH to adapt and form different crystal lattices.

The K1K2 region of Lys-gingipain of Porphyromonas gingivalis blocks induction of HLA expression by gamma interferon.

In the context of periodontal disease, cysteine proteinases or gingipains from Porphyromonas gingivalis have been implicated in the hydrolysis of cytokines, including gamma interferon (IFN-γ). This cytokine plays a crucial role in host defenses, in part, by regulating expression of major histocompatibility complex molecules. Our recent analysis has identified three structurally defined modules, K1, K2, and K3, of the hemagglutinin region of the lysine gingipain Kgp. These three structurally homologous domains have a common ß-sandwich topology that is similar to that found in a superfamily of adhesins and carbohydrate binding domains. The three Kgp hemagglutinin modules are distinguished by variation in some of the loops projecting from the ß-sandwich core. Recombinant products corresponding to both single and multidomain regions as well as native Kgp bound IFN-γ with similar affinities. Among the adhesin domain constructs, only the K1K2 polypeptide inhibited the upregulation of HLA-1 expression in a human erythroleukemia (K562) line induced by both recombinant and native IFN-γ. The K1K2 polypeptide also inhibited HLA-DR expression induced by IFN-γ in human umbilical vein endothelial cells. These effects were competitively inhibited by coincubation with sodium or potassium chloride solution. The N-terminal residues of IFN-γ were implicated in mediating the effect of K1K2, while antibody binding to loop 1 of K2 blocked the action of K1K2. The findings indicate the potential significance of structurally defined Kgp adhesin modules in the inactivation of IFN-γ but also the potential of K1K2 in locating the target for the catalytic domain of Kgp.

The modular structure of haemagglutinin/adhesin regions in gingipains of Porphyromonas gingivalis.

High-molecular-weight arginine- and lysine-specific (Kgp) gingipains are essential virulence factors expressed by the oral pathogen Porphyromonas gingivalis. Haemagglutinin/adhesin (HA) regions of these proteases have been implicated in targeting catalytic domains to biological substrates and in other adhesive functions. We now report the crystal structure of the K3 adhesin domain/module of Kgp, which folds into the distinct ß-jelly roll sandwich topology previously observed for K2. A conserved structural feature of K3, previously observed in the Kgp K2 module, is the half-way point anchoring of the surface exposed loops via an arginine residue found in otherwise highly variable sequences. Small-angle X-ray scattering data for the recombinant construct K1K2K3 confirmed a structure comprising a tandem repeat of three homologous modules, K1, K2 and K3 while also indicating an unusual 'y'-shape arrangement of the modules connected by variable linker sequences. Only the K2 and K3 modules and a K1K2 construct were observed to be potently haemolytic. K2, K3 and the K1K2 construct showed preferential recognition of haem-albumin over albumin whereas only low affinity binding was detected for K1 and the K1K2K3 construct. The data indicate replication of some biological functions over the three adhesin domains of Kgp while other functions are restricted.

Structure and activity of Aspergillus nidulans copper amine oxidase.

Aspergillus nidulans amine oxidase (ANAO) has the unusual ability among the family of copper and trihydroxyphenylalanine quinone-containing amine oxidases of being able to oxidize the amine side chains of lysine residues in large peptides and proteins. We show here that in common with the related enzyme from the yeast Pichia pastoris, ANAO can promote the cross-linking of tropoelastin and oxidize the lysine residues in α-casein proteins and tropoelastin. The crystal structure of ANAO, the first for a fungal enzyme in this family, has been determined to a resolution of 2.4 Å. The enzyme is a dimer with the archetypal fold of a copper-containing amine oxidase. The active site is the most open of any of those of the structurally characterized enzymes in the family and provides a ready explanation for its lysine oxidase-like activity.

Individual differences in sensitivity to violence.

A survey of university students explored correlates of sensitivity to violence, defined as a person's average rating of the severity of violence of several behaviors. The 79 women were somewhat more sensitive than the 39 men. Sensitivity was associated with the tendency to describe oneself as violence sensitive or violence tolerant, with the person's manner of defining the concept of violence, and with the tendency to include verbal abuse and inaction as possible forms of violence. Sensitivity was not clearly associated with some other variables, such as attitudes toward bullying and acceptability of hunting. The potential utility of measuring sensitivity to violence, and further work suggested by these findings, are discussed.

A new crystal form of human vascular adhesion protein 1.

Human vascular adhesion protein 1 (VAP-1) is involved in lymphocyte-endothelial cell adhesion and has been implicated in many human inflammatory diseases. VAP-1 is a member of the copper amine oxidase family of enzymes with a trihydroxyphenylalanine quinone (TPQ) cofactor. Previously characterized crystals of VAP-1 suffered from anisotropy and contained disordered regions; in addition, one form was consistently twinned. In an effort to grow crystals that diffracted to higher resolution for inhibitor-binding studies, a construct with an N-terminal deletion was made and expressed in the Chinese hamster ovary (CHO) glycosylation mutant cell line Lec8. Screening produced crystals that displayed some anisotropy and contained seven molecules per asymmetric unit. These crystals belonged to space group C2, with unit-cell parameters a=394.5, b=115.8, c=179.3 Å, ß=112.3°. The structure was refined to a resolution of 2.9 Å, with Rcryst and Rfree values of 0.250 and 0.286, respectively.

Correlation of active site metal content in human diamine oxidase with trihydroxyphenylalanine quinone cofactor biogenesis .

Copper-containing amine oxidases (CAOs) require a protein-derived topaquinone cofactor (TPQ) for activity. TPQ biogenesis is a self-processing reaction requiring the presence of copper and molecular oxygen. Recombinant human diamine oxidase (hDAO) was heterologously expressed in Drosophila S2 cells, and analysis indicates that the purified hDAO contains substoichiometric amounts of copper and TPQ. The crystal structure of a complex of an inhibitor, aminoguanidine, and hDAO at 2.05 Å resolution shows that the aminoguanidine forms a covalent adduct with the TPQ and that the site is ∼75% occupied. Aminoguanidine is a potent inhibitor of hDAO with an IC(50) of 153 ± 9 nM. The structure indicates that the catalytic metal site, normally occupied by copper, is fully occupied. X-ray diffraction data recorded below the copper edge, between the copper and zinc edges, and above the zinc edge have been used to show that the metal site is occupied approximately 75% by copper and 25% by zinc and the formation of the TPQ cofactor is correlated with copper occupancy.

Sensitivity to violence measured by ratings of severity increases after nonviolence training.

It was hypothesized that training in nonviolence would increase participants' sensitivity to violence because such training emphasizes both the harm and the avoidability of many kinds of violence. This research built upon earlier studies, which had proposed that ratings of the severity of violent behaviors (e.g., murder, bullying, cursing) can be interpreted as measuring sensitivity to violence. Two quasi-experiments examined changes in ratings of severity obtained before and after nonviolence training. In Study 1, 28 college-age traffic offenders who received nonviolence training judged stimulus behaviors ranging from life-threatening physical harm to verbal disrespect as more violent after their training. An untrained comparison group did not show this change. In Study 2, 30 student teachers who received instruction in nonviolence also rated behaviors as more violent after training; an untrained comparison group did not. Results are interpreted as showing increased sensitivity to violence following exposure to nonviolence.

Structure determination and analysis of a haemolytic gingipain adhesin domain from Porphyromonas gingivalis.

Porphyromonas gingivalis is an obligately anaerobic bacterium recognized as an aetiological agent of adult periodontitis. P. gingivalis produces cysteine proteinases, the gingipains. The crystal structure of a domain within the haemagglutinin region of the lysine gingipain (Kgp) is reported here. The domain was named K2 as it is the second of three homologous structural modules in Kgp. The K2 domain structure is a 'jelly-roll' fold with two anti-parallel beta-sheets. This fold topology is shared with adhesive domains from functionally diverse receptors such as MAM domains, ephrin receptor ligand binding domains and a number of carbohydrate binding modules. Possible functions of K2 were investigated. K2 induced haemolysis of erythrocytes in a dose-dependent manner that was augmented by the blocking of anion transport. Further, cysteine-activated arginine gingipain RgpB, which degrades glycophorin A, sensitized erythrocytes to the haemolytic effect of K2. Cleaved K2, similar to that found in extracted Kgp, lacks the haemolytic activity indicating that autolysis of Kgp may be a staged process which is artificially enhanced by extraction of the protein. The data indicate a functional role for K2 in the integrated capacity conferred by Kgp to enable the porphyrin auxotroph P. gingivalis to capture essential haem from erythrocytes.

A new crystal form of human diamine oxidase.

Copper amine oxidases (CAOs) are ubiquitous in nature and catalyse the oxidative deamination of primary amines to the corresponding aldehydes. Humans have three viable CAO genes (AOC1-3). AOC1 encodes human diamine oxidase (hDAO), which is the frontline enzyme for histamine metabolism. hDAO is unique among CAOs in that it has a distinct substrate preference for diamines. The structure of hDAO in space group P2(1)2(1)2(1) with two molecules in the asymmetric unit has recently been reported. Here, the structure of hDAO refined to 2.1 A resolution in space group C222(1) with one molecule in the asymmetric unit is reported.

Structure and inhibition of human diamine oxidase.

Humans have three functioning genes that encode copper-containing amine oxidases. The product of the AOC1 gene is a so-called diamine oxidase (hDAO), named for its substrate preference for diamines, particularly histamine. hDAO has been cloned and expressed in insect cells and the structure of the native enzyme determined by X-ray crystallography to a resolution of 1.8 A. The homodimeric structure has the archetypal amine oxidase fold. Two active sites, one in each subunit, are characterized by the presence of a copper ion and a topaquinone residue formed by the post-translational modification of a tyrosine. Although hDAO shares 37.9% sequence identity with another human copper amine oxidase, semicarbazide sensitive amine oxidase or vascular adhesion protein-1, its substrate binding pocket and entry channel are distinctly different in accord with the different substrate specificities. The structures of two inhibitor complexes of hDAO, berenil and pentamidine, have been refined to resolutions of 2.1 and 2.2 A, respectively. They bind noncovalently in the active-site channel. The inhibitor binding suggests that an aspartic acid residue, conserved in all diamine oxidases but absent from other amine oxidases, is responsible for the diamine specificity by interacting with the second amino group of preferred diamine substrates.

Porphyrin-linked nitroimidazole antibiotics targeting Porphyromonas gingivalis.

Periodontal disease is an inflammatory process affecting supporting tissues surrounding the teeth. The anaerobic gram-negative bacterium Porphyromonas gingivalis is implicated in the disease. This organism requires the uptake of porphyrins most apparently as haem 1 from local haemorrhage and it has a HA2 receptor on the outer membrane for this purpose that provides the opportunity to achieve selective anti-microbial activity. Uniquely, this receptor is based on recognition of porphyrin macrocycle and on a propionic acid side-chain rather than recognition of the coordinated metal ion through chelation, a process used by other organisms with the HasA porphyrin receptor. Porphyrin-antibiotic conjugates 11, 12, 13a and 13b were designed as potential highly selective P. gingivalis inhibitors, a key point being that they are based on the use of free-base porphyrins to render them unpalatable to other organisms. These compounds were synthesised from metronidazole 4 and deuteroporphyrin IX 3. Conjugates 11, 12, 13a and 13b are all recognised by the HA2 receptor of P. gingivalis, bind as strongly as haem 1 to HA2 and are highly effective. For example, the amide-linked mono-metronidazole mono-acid adducts 11 and 12 have the same growth inhibitory activity towards P. gingivalis and both are two-fold more active than metronidazole 4 and ten- to twenty-fold more effective than the metronidazole derivative, amine 5. The methyl esters 9 and 10, in contrast, are not recognised by HA2 and are ineffective in inhibiting P. gingivalis, leading to the conclusion that capture by HA2 may be necessary for activity of the adducts. Preliminary growth inhibition assays involving a range of bacteria have demonstrated the high selectivity of conjugates 13a and 13b towards P. gingivalis.

Sensitive and tolerant raters differ in their magnitude estimates of extremely violent behaviors.

A 2007 study indicated severity of violence of several behaviors can be rated consistently, such that an individual's average rating across the behaviors can serve as a measure of sensitivity to violence. However, violence-sensitive and violence-tolerant raters in that study gave similar high scale ratings of severity of violence for extremely violent behaviors such as murder and stabbing. In the present study an open-ended magnitude estimation response scale was used by 27 participants to examine impressions of the severity of violence of 38 behaviors. There was a positive correlation between estimates of severity for extreme physical violence and estimates for lower severity of violence. This result indicates that the earlier finding of apparent equivalence of sensitive and tolerant individuals did not signify agreement about extremely violent behaviors but was a ceiling effect arising from the closed-ended response scale used. Violence-sensitive and violence-tolerant individuals seem to have different attitudes toward all types of violence.

Structure of N-acetyl-beta-D-glucosaminidase (GcnA) from the endocarditis pathogen Streptococcus gordonii and its complex with the mechanism-based inhibitor NAG-thiazoline.

The crystal structure of GcnA, an N-acetyl-beta-D-glucosaminidase from Streptococcus gordonii, was solved by multiple wavelength anomalous dispersion phasing using crystals of selenomethionine-substituted protein. GcnA is a homodimer with subunits each comprised of three domains. The structure of the C-terminal alpha-helical domain has not been observed previously and forms a large dimerisation interface. The fold of the N-terminal domain is observed in all structurally related glycosidases although its function is unknown. The central domain has a canonical (beta/alpha)(8) TIM-barrel fold which harbours the active site. The primary sequence and structure of this central domain identifies the enzyme as a family 20 glycosidase. Key residues implicated in catalysis have different conformations in two different crystal forms, which probably represent active and inactive conformations of the enzyme. The catalytic mechanism for this class of glycoside hydrolase, where the substrate rather than the enzyme provides the cleavage-inducing nucleophile, has been confirmed by the structure of GcnA complexed with a putative reaction intermediate analogue, N-acetyl-beta-D-glucosamine-thiazoline. The catalytic mechanism is discussed in light of these and other family 20 structures.

Typology of violence derived from ratings of severity and provocation.

Two exploratory studies examined ratings of the severity of violence of several behaviors. In Study 1, a very consistent ordering of the behaviors by severity was obtained from two groups of participants. The stated justification for the behaviors was manipulated, and both mitigation and aggravation effects were observed. Study 2 found that essentially the same ordering of behaviors could be obtained in a provocation-rating task, and that both the severity ratings and the provocation ratings yielded four interpretable types of violence upon factor analysis: more severe physical (V1), less severe physical (V2), more severe nonphysical (V3), and less severe nonphysical (V4). Individual profiles of severity ratings across these four types yielded two interpretable groupings of participants upon cluster analysis: a violence-sensitive group and a violence-tolerant group. The violence-tolerant group had lower severity ratings for three of the four types of violence. These empirical distinctions help to illuminate what appear to be different meanings of the term violent for different behavior categories and for different individuals.