Thiolase: A Versatile Biocatalyst Employing Coenzyme A-Thioester Chemistry for Making and Breaking C-C Bonds.

Thiolases are CoA-dependent enzymes that catalyze the thiolytic cleavage of 3-ketoacyl-CoA, as well as its reverse reaction, which is the thioester-dependent Claisen condensation reaction. Thiolases are dimers or tetramers (dimers of dimers). All thiolases have two reactive cysteines: (a) a nucleophilic cysteine, which forms a covalent intermediate, and (b) an acid/base cysteine. The best characterized thiolase is the Zoogloea ramigera thiolase, which is a bacterial biosynthetic thiolase belonging to the CT-thiolase subfamily. The thiolase active site is also characterized by two oxyanion holes, two active site waters, and four catalytic loops with characteristic amino acid sequence fingerprints. Three thiolase subfamilies can be identified, each characterized by a unique sequence fingerprint for one of their catalytic loops, which causes unique active site properties. Recent insights concerning the thiolase reaction mechanism, as obtained from recent structural studies, as well as from classical and recent enzymological studies, are addressed, and open questions are discussed.

Chryseobacterium metallicongregator, sp. nov., a bacterium possessing metallophore activity towards rare earth elements.

A polyphasic taxonomic study was carried out on strain ES2T, isolated from sediment of a wetland created to remediate acid drainage from a coal mine. The rod-shaped bacterium formed yellow/orange pigmented colonies and produced the pigment flexirubin. The 16S rRNA gene sequence results assigned the strain to Chryseobacterium, with 98.9 and 98.3 % similarity to Chryseobacterium vietnamense and Chryseobacterium cucumeris, respectively. Computation of the average nucleotide identity and digital DNA-DNA hybridization values with the closest phylogenetic neighbours of ES2T revealed genetic differences at the species level, which were further substantiated by differences in several physiological characteristics. The dominant fatty acids of strain ES2T were iso-C15 : 0, iso-C17 : 1 ω9c, iso C17 : 0 3-OH, and iso-C15 : 0 2-OH. The DNA G+C content was 35.5 mol%. The major polar lipid was phosphatidylethanolamine while menaquinone-6 was the only menaquinone found. This bacterium has been previously shown to possess metallophore activity towards rare earth elements, and based on genome sequencing, possesses all required genes for siderophore production/activity, possibly identifying the source of this unique ability. On the basis of the results obtained here, this bacterium is assigned to the genus Chryseobacterium as representing a new species with the name Chryseobacterium metallicongregator sp. nov., type strain ES2T (=NRRL B-65679T=KCTC 102120T).

Goethite and Hematite Nucleation and Growth from Ferrihydrite: Effects of Oxyanion Surface Complexes.

The presence of oxyanions, such as nitrate (NO3-) and phosphate (PO43-), regulates the nucleation and growth of goethite (Gt) and hematite (Hm) during the transformation of ferrihydrite (Fh). Our previous studies showed that oxyanion surface complexes control the rate and pathway of Fh transformation to Gt and Hm. However, how oxyanion surface complexes control the mechanism of Gt and Hm nucleation and growth during the Fh transformation is still unclear. We used synchrotron scattering methods and cryogenic transmission electron microscopy to investigate the effects of NO3- outer-sphere complexes and PO43- inner-sphere complexes on the mechanism of Gt and Hm formation from Fh. Our TEM results indicated that Gt particles form through a two-step model in which Fh particles first transform to Gt nanoparticles and then crystallographically align and grow to larger particles by oriented attachment (OA). In contrast, for the formation of Hm, imaging shows that Fh particles first aggregate and then transform to Hm through interface nucleation. This is consistent with our X-ray scattering results, which demonstrate that NO3- outer-sphere and PO43- inner-sphere complexes promote the formation of Gt and Hm, respectively. These results have implications for understanding the coupled interactions of oxyanions and iron oxy-hydroxides in Earth-surface environments.

Conserved loop residues-Tyr270 and Asn372 near the catalytic site of the lysostaphin endopeptidase are essential for staphylolytic activity toward pentaglycine binding and catalysis.

Lysostaphin endopeptidase cleaves pentaglycine cross-bridges found in staphylococcal cell-wall peptidoglycans and proves very effective in combatting methicillin-resistant Staphylococcus aureus. Here, we revealed the functional importance of two loop residues, Tyr270 in loop 1 and Asn372 in loop 4, which are highly conserved among the M23 endopeptidase family and are found close to the Zn2+-coordinating active site. Detailed analyses of the binding groove architecture together with protein-ligand docking showed that these two loop residues potentially interact with the docked ligand-pentaglycine. Ala-substituted mutants (Y270A and N372A) were generated and over-expressed in Escherichia coli as a soluble form at levels comparable to the wild type. A drastic decrease in staphylolytic activity against S. aureus was observed for both mutants, suggesting an essential role of the two loop residues in lysostaphin function. Further substitutions with an uncharged polar Gln side-chain revealed that only the Y270Q mutation caused a dramatic reduction in bioactivity. In silico predicting the effect of binding site mutations revealed that all mutations displayed a large ΔΔGbind value, signifying requirements of the two loop residues for efficient binding to pentaglycine. Additionally, MD simulations revealed that Y270A and Y270Q mutations induced large flexibility of the loop 1 region, showing markedly increased RMSF values. Further structural analysis suggested that Tyr270 conceivably participated in the oxyanion stabilization of the enzyme catalysis. Altogether, our present study disclosed that two highly conserved loop residues, loop 1-Tyr270 and loop 4-Asn372, located near the lysostaphin active site are crucially involved in staphylolytic activity toward binding and catalysis of pentaglycine cross-links.

Gordonia metallireducens sp. nov., a tellurite- and selenite-resistant bacterium isolated from the sediment of an acid mine drainage stream.

A polyphasic taxonomic study was carried out on strain TSed Te1T, isolated from sediment of a stream contaminated with acid drainage from a coal mine. The bacterium forms pink-pigmented colonies and has a rod-coccus growth cycle, which also includes some coryneform arrangements. This bacterium is capable of growing in the presence of up to 750 µg ml-1 tellurite and 5000 µg ml-1 selenite, reducing each to elemental form. Nearly complete 16S rRNA gene sequence analysis associated the strain with Gordonia, with 99.5 and 99.3 % similarity to Gordonia namibiensis and Gordonia rubripertincta, respectively. Computation of the average nucleotide identity and digital DNA-DNA hybridization comparisons with the closest phylogenetic neighbour of TSed Te1T revealed genetic differences at the species level, which were further substantiated by differences in several physiological characteristics. The dominant fatty acids were C16 : 0, C18 : 1, C16 : 1 and tuberculostearic acid. The DNA G+C content was 67.6 mol%. Major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside, while MK-9(H2) was the only menaquinone found. Mycolic acids of C56-C60 were present. Whole-cell hydrolysates contained meso-diaminopimelic acid along with arabinose and galactose as the major cell-wall sugars. On the basis of the results obtained in this study, the bacterium was assigned to the genus Gordonia and represents a new species with the name Gordonia metallireducens sp. nov. The type strain is TSed Te1T (=NRRL B-65678T=DSM 114093T).

In Situ Regulation of MnO2 Structural Characteristics by Oxyanions to Boost Permanganate Autocatalysis for Phenol Removal.

Oxyanions, a class of constituents naturally occurring in water, have been widely demonstrated to enhance permanganate (Mn(VII)) decontamination efficiency. However, the detailed mechanism remains ambiguous, mainly because the role of oxyanions in regulating the structural parameters of colloidal MnO2 to control the autocatalytic activity of Mn(VII) has received little attention. Herein, the origin of oxyanion-induced enhancement is systematically studied using theoretical calculations, electrochemical tests, and structure-activity relation analysis. Using bicarbonate (HCO3-) as an example, the results indicate that HCO3- can accelerate the degradation of phenol by Mn(VII) by improving its autocatalytic process. Specifically, HCO3- plays a significant role in regulating the structure of in situ produced MnO2 colloids, i.e., increasing the surface Mn(III)s content and restricting particle growth. These structural changes in MnO2 facilitate its strong binding to Mn(VII), thereby triggering interfacial electron transfer. The resultant surface-activated Mn(VII)* complexes demonstrate excellent degrading activity via directly seizing one electron from phenol. Further, other oxyanions with appropriate ionic potentials (i.e., borate, acetate, metasilicate, molybdate, and phosphate) exhibit favorable influences on the oxidative capability of Mn(VII) through an activation mechanism similar to that of HCO3-. These findings considerably improve our fundamental understanding of the oxidation behavior of Mn(VII) in actual water environments and provide a theoretical foundation for designing autocatalytically boosted Mn(VII) oxidation systems.

Toxic heavy metal/oxyanion tolerance in haloarchaea from some saline and hypersaline ecosystems.

Toxic heavy metal/oxyanion contamination has increased severely through the last decades. In this study, 169 native haloarchaeal strains were isolated from different saline and hypersaline econiches of Iran. After providing pure culture and performing morphological, physiological, and biochemical tests, haloarchaea resistance toward arsenate, selenite, chromate, cadmium, zinc, lead, copper, and mercury were surveyed using an agar dilution method. On the basis of minimum inhibitory concentrations (MICs), the least toxicities were found with selenite and arsenate, while the haloarchaeal strains revealed the highest sensitivity for mercury. On the other hand, the majority of haloarchaeal strains exhibited similar responses to chromate and zinc, whereas the resistance level of the isolates to lead, cadmium, and copper was very heterogeneous. 16 S ribosomal RNA (rRNA) gene sequence analysis revealed that most haloarchaeal strains belong to the Halorubrum and Natrinema genera. The obtained results from this study showed that among the identified isolates, Halococcus morrhuae strain 498 had an exceptional resistance toward selenite and cadmium (64 and 16 mM, respectively). Also, Halovarius luteus strain DA5 exhibited a remarkable tolerance against copper (32 mM). Moreover, strain Salt5, identified as Haloarcula sp., was the only strain that could tolerate all eight tested heavy metals/oxyanions and had a significant tolerance of mercury (1.5 mM).

Oxyanion Surface Complexes Control the Kinetics and Pathway of Ferrihydrite Transformation to Goethite and Hematite.

The rate and pathway of ferrihydrite (Fh) transformation at oxic conditions to more stable products is controlled largely by temperature, pH, and the presence of other ions in the system such as nitrate (NO3-), sulfate (SO42-), and arsenate (AsO43-). Although the mechanism of Fh transformation and oxyanion complexation have been separately studied, the effect of surface complex type and strength on the rate and pathway remains only partly understood. We have developed a kinetic model that describes the effects of surface complex type and strength on Fh transformation to goethite (Gt) and hematite (Hm). Two sets of oxyanion-adsorbed Fh samples were prepared, nonbuffered and buffered, aged at 70 ± 1.5 °C, and then characterized using synchrotron X-ray scattering methods and wet chemical analysis. Kinetic modeling showed a significant decrease in the rate of Fh transformation for oxyanion surface complexes dominated by strong inner-sphere (SO42- and AsO43-) versus weak outer-sphere (NO3-) bonding and the control. The results also showed that the Fh transformation pathway is influenced by the type of surface complex such that with increasing strength of bonding, a smaller fraction of Gt forms compared with Hm. These findings are important for understanding and predicting the role of Fh in controlling the transport and fate of metal and metalloid oxyanions in natural and applied systems.

Tellurite and Selenite: how can these two oxyanions be chemically different yet so similar in the way they are transformed to their metal forms by bacteria?

This opinion review explores the microbiology of tellurite, TeO32- and selenite, SeO32- oxyanions, two similar Group 16 chalcogen elements, but with slightly different physicochemical properties that lead to intriguing biological differences. Selenium, Se, is a required trace element compared to tellurium, Te, which is not. Here, the challenges around understanding the uptake transport mechanisms of these anions, as reflected in the model organisms used by different groups, are described. This leads to a discussion around how these oxyanions are subsequently reduced to nanomaterials, which mechanistically, has controversies between ideas around the molecule chemistry, chemical reactions involving reduced glutathione and reactive oxygen species (ROS) production along with the bioenergetics at the membrane versus the cytoplasm. Of particular interest is the linkage of glutathione and thioredoxin chemistry from the cytoplasm through the membrane electron transport chain (ETC) system/quinones to the periplasm. Throughout the opinion review we identify open and unanswered questions about the microbial physiology under selenite and tellurite exposure. Thus, demonstrating how far we have come, yet the exciting research directions that are still possible. The review is written in a conversational manner from three long-term researchers in the field, through which to play homage to the late Professor Claudio Vásquez.

Quantum Biochemistry and MM-PBSA Description of the ZIKV NS2B-NS3 Protease: Insights into the Binding Interactions beyond the Catalytic Triad Pocket.

The Zika virus protease NS2B-NS3 has a binding site formed with the participation of a H51-D75-S135 triad presenting two forms, active and inactive. Studies suggest that the inactive conformation is a good target for the design of inhibitors. In this paper, we evaluated the co-crystallized structures of the protease with the inhibitors benzoic acid (5YOD) and benzimidazole-1-ylmethanol (5H4I). We applied a protocol consisting of two steps: first, classical molecular mechanics energy minimization followed by classical molecular dynamics were performed, obtaining stabilized molecular geometries; second, the optimized/relaxed geometries were used in quantum biochemistry and molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) calculations to estimate the ligand interactions with each amino acid residue of the binding pocket. We show that the quantum-level results identified essential residues for the stabilization of the 5YOD and 5H4I complexes after classical energy minimization, matching previously published experimental data. The same success, however, was not observed for the MM-PBSA simulations. The application of quantum biochemistry methods seems to be more promising for the design of novel inhibitors acting on NS2B-NS3.

Transesterification of Non-Activated Esters Promoted by Small Molecules Mimicking the Active Site of Hydrolases.

The synthesis of small molecules able to mimic the active site of hydrolytic enzymes has been largely pursued in recent decades. The high reaction rates and specificity shown by natural hydrolases present an attractive target, and yet the preparation of suitable small-molecule mimics remains challenging, requiring activated substrates to achieve productive outcomes. Here we present small synthetic artificial enzymes which mimic the catalytic site and the oxyanion hole of chymotrypsin and N-terminal hydrolases and are able to perform, for the first time, the transesterification of a non-activated ester such as ethyl acetate with methanol under mild and neutral reaction conditions.

Mutations in penicillin-binding protein 2 from cephalosporin-resistant Neisseria gonorrhoeae hinder ceftriaxone acylation by restricting protein dynamics.

The global incidence of the sexually transmitted disease gonorrhea is expected to rise due to the spread of Neisseria gonorrhoeae strains with decreased susceptibility to extended-spectrum cephalosporins (ESCs). ESC resistance is conferred by mosaic variants of penicillin-binding protein 2 (PBP2) that have diminished capacity to form acylated adducts with cephalosporins. To elucidate the molecular mechanisms of ESC resistance, we conducted a biochemical and high-resolution structural analysis of PBP2 variants derived from the decreased-susceptibility N. gonorrhoeae strain 35/02 and ESC-resistant strain H041. Our data reveal that mutations both lower affinity of PBP2 for ceftriaxone and restrict conformational changes that normally accompany acylation. Specifically, we observe that a G545S substitution hinders rotation of the ß3 strand necessary to form the oxyanion hole for acylation and also traps ceftriaxone in a noncanonical configuration. In addition, F504L and N512Y substitutions appear to prevent bending of the ß3-ß4 loop that is required to contact the R1 group of ceftriaxone in the active site. Other mutations also appear to act by reducing flexibility in the protein. Overall, our findings reveal that restriction of protein dynamics in PBP2 underpins the ESC resistance of N. gonorrhoeae.

An Adjustable Cleft Based on an 8-Sulfonamide-2-Naphthoic Acid with Oxyanion Hole Geometry.

Cleft type receptors showing the oxyanion hole motif have been prepared in a straightforward synthesis starting from the commercial 3,7-dihidroxy-2-naphthoic acid. The double H-bond donor pattern is achieved by the introduction of a sulfonamide group in the C-8 position of naphthalene and a carboxamide at the C-2 position. This cleft, for which the geometry resembles that of an oxyanion hole, is able to adjust to different guests, as shown by the analysis of the X-ray crystal structures of associates with methanol or acetic acid. Combination of hydrogen bonds and charge-transfer interactions led to further stabilization of the complexes, in which the electron-rich aromatic ring of the receptor was close in space to the electron-deficient dinitroaromatic guests. Modelling studies and bidimensional NMR experiments have been carried out to provide additional information.

A Cd(II) Luminescent Coordination Grid as a Multiresponsive Fluorescence Sensor for Cr(VI) Oxyanions and Cr(III), Fe(III), and Al(III) in Aqueous Medium.

Hydro(solvo)thermal reactions of Cd(NO3)2, N-(pyridin-3-ylmethyl)-4-(pyridin-4-yl)-1,8-naphthalimide (NI-mbpy-34), and 5-bromobenzene-1,3-dicarboxylic acid (Br-1,3-H2bdc) afforded a luminescent coordination polymer, {[Cd(Br-1,3-bdc)(NI-mbpy-34)(H2O)]∙2H2O}n (1). Single-crystal X-ray diffraction analysis showed that 1 features a two-dimensional (2-D) gridlike sql layer with the point symbol of (44·62), where the Cd(II) center adopts a {CdO5N2} pentagonal bipyramidal geometry. Thermogravimetric (TG) analysis confirmed the thermal stability of 1 up to about 340 °C, whereas XRPD patterns proved the maintenance of crystallinity and framework integrity of 1 in CH2Cl2, H2O, CH3OH, and toluene. Photoluminescence studies indicated that 1 displayed intense blue fluorescence emissions in both solid-state and H2O suspension-phase. Owing to the good fluorescent properties, 1 could serve as an excellent turn-off fluorescence sensor for selective and sensitive Cr(VI) detection in water, with LOD = 15.15 µM for CrO42- and 14.91 µM for Cr2O72-, through energy competition absorption mechanism. In addition, 1 could also sensitively detect Cr3+, Fe3+, and Al3+ ions in aqueous medium via fluorescence-enhancement responses, with LOD = 2.81 µM for Cr3+, 3.82 µM for Fe3+, and 3.37 µM for Al3+, mainly through an absorbance-caused enhancement (ACE) mechanism.

Mesoporous Poly(melamine-co-formaldehyde) Particles for Efficient and Selective Phosphate and Sulfate Removal.

Due to the existence-threatening risk to aquatic life and entire ecosystems, the removal of oxyanions such as sulfate and phosphate from anthropogenic wastewaters, such as municipal effluents and acid mine drainage, is inevitable. Furthermore, phosphorus is an indispensable resource for worldwide plant fertilization, which cannot be replaced by any other substance. This raises phosphate to one of the most important mineral resources worldwide. Thus, efficient recovery of phosphate is essential for ecosystems and the economy. To face the harsh acidic conditions, such as for acid mine drainage, an adsorber material with a high chemical resistivity is beneficial. Poly(melamine-co-formaldehyde) (PMF) sustains these conditions whilst its very high amount of nitrogen functionalities (up to 53.7 wt.%) act as efficient adsorption sides. To increase adsorption capacities, PMF was synthesized in the form of mesoporous particles using a hard-templating approach yielding specific surface areas up to 409 m2/g. Different amounts of silica nanospheres were utilized as template and evaluated for the adsorption of sulfate and phosphate ions. The adsorption isotherms were validated by the Langmuir model. Due to their properties, the PMF particles possessed outperforming maximum adsorption capacities of 341 and 251 mg/g for phosphate and sulfate, respectively. Furthermore, selective adsorption of sulfate from mixed solutions of phosphate and sulfate was found for silica/PMF hybrid particles.

MD simulations reveal alternate conformations of the oxyanion hole in the Zika virus NS2B/NS3 protease.

Recent crystallography studies have shown that the binding site oxyanion hole plays an important role in inhibitor binding, but can exist in two conformations (active/inactive). We have undertaken molecular dynamics (MD) calculations to better understand oxyanion hole dynamics and thermodynamics. We find that the Zika virus (ZIKV) NS2B/NS3 protease maintains a stable closed conformation over multiple 100-ns conventional MD simulations in both the presence and absence of inhibitors. The S1, S2, and S3 pockets are stable as well. However, in two of eight simulations, the A132-G133 peptide bond in the binding pocket of S1' spontaneously flips to form a 310 -helix that corresponds to the inactive conformation of the oxyanion hole, and then maintains this conformation until the end of the 100-ns conventional MD simulations without inversion of the flip. This conformational change affects the S1' pocket in ZIKV NS2B/NS3 protease active site, which is important for small molecule binding. The simulation results provide evidence at the atomic level that the inactive conformation of the oxyanion hole is more favored energetically when no specific interactions are formed between substrate/inhibitor and oxyanion hole residues. Interestingly, however, transition between the active and inactive conformation of the oxyanion hole can be observed by boosting the valley potential in accelerated MD simulations. This supports a proposed induced-fit mechanism of ZIKV NS2B/NS3 protease from computational methods and provides useful direction to enhance inhibitor binding predictions in structure-based drug design.

An Oxy-anion Accelerated [1,5]-o-Quinone Methide Shift During the Nucleophilic Epoxidation of Salicylfulvene.

A new facet of nucleophilic fulvene epoxidations has been uncovered. 6-Arylfulvenes containing an ortho or para hydroxyl group react with basic hydrogen peroxide in an unusual manner; the epoxidation of the fulvene exocyclic double bond is followed by a phenoxide ion initiated epoxide ring opening to form an o-quinone methide (o-QM) intermediate. The resulting cyclopentadienolate undergoes an unusual oxy-anion accelerated [1,5]-sigmatropic o-QM shift. Computational studies reveal that the activation energy for the [1,5]-QM-shift in the cyclopentadienolate intermediate is quite low, signifying the acceleration caused by the oxy-anion group. Placement of a second hydroxyl group in the 6-aryl ring at C5 epoxidation via electron donation to the o-QM carbon; instead, an intramolecular oxa-6-π-electrocyclization of the o-QM intermediate onto the cyclopentadiene is observed.

Colonization and degradation of polyhydroxyalkanoates by lipase-producing bacteria.

Biodegradation of short-chain-length polyhydroxyalkanoates (scl-PHAs) and medium-chain-length polyhydroxyalkanoates (mcl-PHAs) was studied using 2 bacteria, Pseudomonas chlororaphis and Acinetobacter lwoffii, which secrete an enzyme, or enzymes, with lipase activity. These bacteria produced clear zones of depolymerization on Petri plates containing colloidal solutions of PHA polymers with different monomer compositions. Lipase activity in these bacteria was measured using p-nitrophenyl octanoate as a substrate. In liquid medium, scl-PHA (e.g., PHBV) and mcl-PHA (e.g., PHO) films were used as the sole carbon source for growth, and after 7 days, 5%-18% loss in mass of PHA films was observed. Scanning electron microscopy of these films revealed bacterial colonization of the polymers, with cracks and pitting in the film surfaces. Degradation of polymers released 3-hydroxyhexanoate, 3-hydroxyoctanoate, and 3-hydroxydecanoate monomers into the liquid medium, depending on the starting polymer. Genes encoding secretory lipases, with amino acid consensus sequences for lipase boxes and oxyanion holes, were identified in the genomes of P. chlororaphis and A. lwoffii. Although amino acid consensus sequences for lipase boxes and oxyanion holes are also present in PHA depolymerases identified in the genomes of other PHA-degrading bacteria, the P. chlororaphis and A. lwoffii lipases had low homology with these depolymerases.

A Schiff base modified graphene oxide film for anodic stripping voltammetric determination of arsenite.

A protocol is described for chemical modification of graphene oxide with a Schiff base derived from diethylenetriamine and 2-hydroxy-4-methoxybenzophenone. The base was grafted onto an indium tin oxide (ITO) film and applied to electroanalytical determination of arsenite. Successful grafting was confirmed by Fourier transform-infrared spectroscopy, spectrophotometry, field emission scanning electron microscopy and cyclic voltammetry. Secondly, the coated ITO film served as a working electrode for the stripping voltammetric determination of arsenite. The analytical signal is generated by selective oxidation of metal species via multi-donor sites present in the derivatized Schiff base. The electroanalytical protocol was optimized by investigating the effects of deposition time, working potential, frequency and amplitude of square wave anodic stripping voltammetry. The method has attractive features including (a) the usage of a non-metallic, non-toxic and cost-effective material; (b) improved sensitivity (with limit of detection as low as 156 pM) due to better adsorption of arsenite in the Schiff base pockets on the ITO, and (c) the application to the determination of arsenite in real samples. Graphical abstract Schematic representation of the fabrication of a Schiff base-functionalized graphene oxide on an indium tin oxide (SB@SiO2@GO@ITO) electrode for selective electrochemical sensing of arsenite due to adsorption on multi-donor sites.

Human γ-Glutamyl Transpeptidase 1: STRUCTURES OF THE FREE ENZYME, INHIBITOR-BOUND TETRAHEDRAL TRANSITION STATES, AND GLUTAMATE-BOUND ENZYME REVEAL NOVEL MOVEMENT WITHIN THE ACTIVE SITE DURING CATALYSIS.

γ-Glutamyl transpeptidase 1 (GGT1) is a cell surface, N-terminal nucleophile hydrolase that cleaves glutathione and other γ-glutamyl compounds. GGT1 expression is essential in cysteine homeostasis, and its induction has been implicated in the pathology of asthma, reperfusion injury, and cancer. In this study, we report four new crystal structures of human GGT1 (hGGT1) that show conformational changes within the active site as the enzyme progresses from the free enzyme to inhibitor-bound tetrahedral transition states and finally to the glutamate-bound structure prior to the release of this final product of the reaction. The structure of the apoenzyme shows flexibility within the active site. The serine-borate-bound hGGT1 crystal structure demonstrates that serine-borate occupies the active site of the enzyme, resulting in an enzyme-inhibitor complex that replicates the enzyme's tetrahedral intermediate/transition state. The structure of GGsTop-bound hGGT1 reveals its interactions with the enzyme and why neutral phosphonate diesters are more potent inhibitors than monoanionic phosphonates. These structures are the first structures for any eukaryotic GGT that include a molecule in the active site covalently bound to the catalytic Thr-381. The glutamate-bound structure shows the conformation of the enzyme prior to release of the final product and reveals novel information regarding the displacement of the main chain atoms that form the oxyanion hole and movement of the lid loop region when the active site is occupied. These data provide new insights into the mechanism of hGGT1-catalyzed reactions and will be invaluable in the development of new classes of hGGT1 inhibitors for therapeutic use.