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Tissue factor (TF) is a membrane protein involved in blood coagulation. TF initiates a cascade of proteolytic reactions, ultimately leading to the formation of a blood clot. The first reaction consists of the binding of the coagulation factor VII and its conversion to the activated form, FVIIa. Here, we combined experimental, i.e. quartz crystal microbalance with dissipation monitoring and neutron reflectometry, and computational, i.e. molecular dynamics (MD) simulation, methods to derive a complete structural model of TF and TF/FVIIa complex in a lipid bilayer. This model shows that the TF transmembrane domain (TMD), and the flexible linker connecting the TMD to the extracellular domain (ECD), define the location of the ECD on the membrane surface. The average orientation of the ECD relative to the bilayer surface is slightly tilted towards the lipid headgroups, a conformation that we suggest is promoted by phosphatidylserine lipids, and favours the binding of FVIIa. On the other hand, the formation of the TF/FVIIa complex induces minor changes in the TF structure, and reduces the conformational freedom of both TF and FVIIA. Altogether we describe the protein-protein and protein-lipid interactions favouring blood coagulation, but also instrumental to the development of new drugs. (C) 2022 Elsevier Inc. All rights reserved.

Lytic Polysaccharide Monooxygenases (LPMOs) oxidatively cleave recalcitrant polysaccharides. The mechanism involves (i) reduction of the Cu, (ii) polysaccharide binding, (iii) binding of different oxygen species, and (iv) glycosidic bond cleavage. However, the complete mechanism is poorly understood and may vary across different families and even within the same family. Here, we have investigated the protonation state of a secondary co-ordination sphere histidine, conserved across AA9 family LPMOs that has previously been proposed to be a potential proton donor. Partial unrestrained refinement of newly obtained higher resolution data for two AA9 LPMOs and re-refinement of four additional data sets deposited in the PDB were carried out, where the His was refined without restraints, followed by measurements of the His ring geometrical parameters. This allowed reliable assignment of the protonation state, as also validated by following the same procedure for the His brace, for which the protonation state is predictable. The study shows that this histidine is generally singly protonated at the N epsilon 2 atom, which is close to the oxygen species binding site. Our results indicate robustness of the method. In view of this and other emerging evidence, a role as proton donor during catalysis is unlikely for this His.

Lytic Polysaccharide Monooxygenases (LPMOs) oxidatively cleave recalcitrant polysaccharides. The mechanism involves (i) reduction of the Cu, (ii) polysaccharide binding, (iii) binding of different oxygen species, and (iv) glycosidic bond cleavage. However, the complete mechanism is poorly understood and may vary across different families and even within the same family. Here, we have investigated the protonation state of a secondary co-ordination sphere histidine, conserved across AA9 family LPMOs that has previously been proposed to be a potential proton donor. Partial unrestrained refinement of newly obtained higher resolution data for two AA9 LPMOs and re-refinement of four additional data sets deposited in the PDB were carried out, where the His was refined without restraints, followed by measurements of the His ring geometrical parameters. This allowed reliable assignment of the protonation state, as also validated by following the same procedure for the His brace, for which the protonation state is predictable. The study shows that this histidine is generally singly protonated at the N epsilon 2 atom, which is close to the oxygen species binding site. Our results indicate robustness of the method. In view of this and other emerging evidence, a role as proton donor during catalysis is unlikely for this His.

Lytic polysaccharide monooxygenases (LPMOs) are Cu-containing enzymes that facilitate the degradation of recalcitrant polysaccharides by the oxidative cleavage of glycosidic bonds. They are gaining rapidly increasing attention as key players in biomass conversion, especially for the production of second-generation biofuels. Elucidation of the detailed mechanism of the LPMO reaction is a major step toward the assessment and optimization of LPMO efficacy in industrial biotechnology, paving the way to utilization of sustainable fuel sources. Here, we used density functional theory calculations to study the reaction pathways suggested to date, exploiting a very large active-site model for a fungal AA9 LPMO and using a celloheptaose unit as a substrate mimic. We identify a copper oxyl intermediate as being responsible for H-atom abstraction from the substrate, followed by a rapid, water-assisted hydroxyl rebound, leading to substrate hydroxylation.

A main reason for catalyst deactivation in supported catalysts for methanol synthesis is copper particle growth. We have functionalized the support surface in order to suppress the formation and/or transport of mobile copper species and thereby catalyst deactivation. A Stober silica support was functionalized by treatment with aminopropyltriethoxysilane, which introduces aminopropyl groups on the surface. Copper was deposited on both unfunctionalized and functionalized Stober silica via incipient wetness impregnation with aqueous copper nitrate solutions followed by drying and calcination. Similar particle size distributions (1-5 nm) were obtained for both supports by changing the flow of N-2 to a flow of 2% NO/N-2 during calcination of the unfunctionalized and amine-functionalized silica, respectively. The effect of support functionalization with aminopropyl groups was an increased stability in the methanol synthesis reaction (40 bar, 260 degrees C, 23% CO/7% CO2/60% H-2/10% Ar, 3% COx conversion) due to more limited copper particle growth as determined by transmission electron microscopy (TEM). Changing the interparticle distance did not have an influence on the deactivation rate, while the addition of few very large copper particles did, indicating that Ostwald ripening was most probably the dominant particle growth mechanism for these samples. In situ TEM images showed the contact angle between the reduced copper particles and the support. As shape and size was similar on silica as on amine-functionalized silica, the thermodynamic stability of the copper particles was unaltered. The driving force for copper particle growth was thus unchanged upon functionalization. We therefore suggest that Ostwald ripening in methanol synthesis catalysts was retarded by inhibiting the transport of copper species over the support surface. Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) revealed a decrease in the number of surface groups (hydroxyl, methoxy

A diiron dithiolate complex with a pendant phosphine coordinated to one of the iron centers, [(μ-SCH2)2N(CH2C 6H4-o-PPh2){Fe2(CO)5}] (1), was prepared and structurally characterized. The pendant phosphine is dissociated together with a CO ligand in the presence of excess PMe3, to afford [(μ-SCH2)2N(CH2C 6H4-o-PPh2){Fe(CO)2(PMe 3)}2] (2). Redox reactions of 2 and related complexes were studied in detail by in situ IR spectroscopy. A series of new Fe IIFeI ([3]+ and [6]+), Fe IIFeII ([4]2+), and FeIFe I (5) complexes relevant to Hox, Hox CO, and Hred states of the [FeFe]-hydrogenase active site were detected. Among these complexes, the molecular structures of the diferrous complex [4]2+ with the internal amine and the pendant phosphine co-coordinated to the same iron center and the triphosphine diiron complex 5 were determined by X-ray crystallography. To make a comparison, the redox reactions of an analogous complex, [(μ-SCH2)2N(CH 2C6H5){Fe(CO)2(PMe 3)}2] (7), were also investigated by in situ IR spectroscopy in the absence or presence of extrinsic PPh3, which has no influence on the oxidation reaction of 7. The pendant phosphine in the second coordination sphere makes the redox reaction of 2 different from that of its analogue 7., QC 20140312

Acetate kinase (ACK) (EC no: 2.7.2.1) interconverts acetyl-phosphate and acetate to either catabolize or synthesize acetyl-CoA dependent on the metabolic requirement. Among all ACK entries available in UniProt, we found that around 45% are multiple ACKs in some organisms including more than 300 species but surprisingly, little work has been done to clarify whether this has any significance. In an attempt to gain further insight we have studied the two ACKs (AckA1, AckA2) encoded by two neighboring genes conserved in Lactococcus lactis (L. lactis) by analyzing protein sequences, characterizing transcription structure, determining enzyme characteristics and effect on growth physiology. The results show that the two ACKs are most likely individually transcribed. AckA1 has a much higher turnover number and AckA2 has a much higher affinity for acetate in vitro. Consistently, growth experiments of mutant strains reveal that AckA1 has a higher capacity for acetate production which allows faster growth in an environment with high acetate concentration. Meanwhile, AckA2 is important for fast acetate-dependent growth at low concentration of acetate. The results demonstrate that the two ACKs have complementary physiological roles in L. lactis to maintain a robust acetate metabolism for fast growth at different extracellular acetate concentrations. The existence of ACK isozymes may reflect a common evolutionary strategy in bacteria in an environment with varying concentrations of acetate.

[FeFe]-hydrogenases are enzymes in nature that catalyze the reduction of protons and the oxidation of H-2 at neutral pH with remarkably high activities and incredibly low overpotential. Structural and functional biomimicking of the active site of [FeFe]-hydrogenases can provide helpful hints for elucidating the mechanism of H-2 evolution and uptake at the [FeFe]-hydrogenase active site and for designing bio-inspired catalysts to replace the expensive noble metal catalysts for H-2 generation and uptake. This perspective focuses on the recent progress in the formation and reactivity of iron hydrides closely related to the processes of proton reduction and hydrogen oxidation mediated by diiron dithiolate complexes. The second section surveys the bridging and terminal hydride species formed from various diiron complexes as well as the intramolecular proton transfer. The very recent progress in H-2 activation by diiron dithiolate models are reviewed in the third section. In the concluding remarks and outlook, the differences in structure and catalytic mechanism between the synthetic models and the native [FeFe]-H(2)ase active site are compared and analyzed, which may cause the need for a significantly larger driving force and may lead to lower activities of synthetic models than the [FeFe]-H(2)ases for H-2 generation and uptake., QC 20130906

Pyranose dehydrogenases (PDHs) are extracellular flavin-dependent oxidoreductases secreted by litter-decomposing fungi with a role in natural recycling of plant matter. All major monosaccharides in lignocellulose are oxidized by PDH at comparable yields and efficiencies. Oxidation takes place as single-oxidation or sequential double-oxidation reactions of the carbohydrates, resulting in sugar derivatives oxidized primarily at C2, C3 or C2/3 with the concomitant reduction of the flavin. A suitable electron acceptor then reoxidizes the reduced flavin. Whereas oxygen is a poor electron acceptor for PDH, several alternative acceptors, e.g., quinone compounds, naturally present during lignocellulose degradation, can be used. We have determined the 1.6-Å crystal structure of PDH from Agaricus meleagris. Interestingly, the flavin ring in PDH is modified by a covalent mono- or di-atomic species at the C(4a) position. Under normal conditions, PDH is not oxidized by oxygen; however, the related enzyme pyranose 2-oxidase (P2O) activates oxygen by a mechanism that proceeds via a covalent flavin C(4a)-hydroperoxide intermediate. Although the flavin C(4a) adduct is common in monooxygenases, it is unusual for flavoprotein oxidases, and it has been proposed that formation of the intermediate would be unfavorable in these oxidases. Thus, the flavin adduct in PDH not only shows that the adduct can be favorably accommodated in the active site, but also provides important details regarding the structural, spatial and physicochemical requirements for formation of this flavin intermediate in related oxidases. Extensive in silico modeling of carbohydrates in the PDH active site allowed us to rationalize the previously reported patterns of substrate specificity and regioselectivity. To evaluate the regioselectivity of D-glucose oxidation, reduction experiments were performed using fluorinated glucose. PDH was rapidly reduced by 3-fluorinated glucose, which has the C2 position accessible fo, QC 20130213

A general question in biochemistry is the interplay between the chemical properties of cofactors and the surrounding protein matrix. Here, the functions of NADP(+) and FAD are explored by investigation of a representative monooxygenase reconstituted with chemically modified cofactor analogues. Like pieces of a jigsaw puzzle, the enzyme active site juxtaposes the flavin and nicotinamide rings, harnessing their H-bonding and steric properties to finely construct an oxygen-reacting center that restrains the flavin peroxide intermediate in a catalytically competent orientation. Strikingly, the regio- and stereoselectivities of the reaction are essentially unaffected by cofactor modifications. These observations indicate a remarkable robustness of this complex multicofactor active site, which has implications for enzyme design based on cofactor engineering approaches.

A lucky break: A combination of steric pressure and electron asymmetry has provided the first example of a diiron dithiolate that is both rotated and basic. The present work establishes the feasibility of a hydride free rotated structure for the Hred state of the enzyme (see figure). Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The transport of small ligands to active sites of proteins is the basis of vital processes in biology such as enzymatic catalysis and cell signaling, but also of more destructive ones including enzyme inhibition and oxidative damage. Here, we show how a diffusion-reaction model solved by means of molecular dynamics and density functional theory calculations provides novel insight into the transport of small ligands in proteins. In particular, we unravel the existence of an elusive, dynamically formed gas channel, which CO2 takes to diffuse from the solvent to the active site (C-cluster) of the bifunctional multisubunit enzyme complex carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS). Two cavities forming this channel are temporarily created by protein fluctuations and are not apparent in the X-ray structures. The ligand transport is controlled by two residues at the end of this tunnel, His113 and His 116, and occurs on the same time scale on which chemical binding to the active site takes place (0.1-1 ms), resulting in an overall binding rate on the second time scale. We find that upon reduction of CO2 to CO, the newly formed Fe-hydroxy ligand greatly strengthens the hydrogen-bond network, preventing CO from exiting the protein through the same way that CO2 takes to enter the protein. This is the basis for directional transport of CO from the production site (C-cluster of CODH subunit) to the utilization site (A-cluster of ACS subunit). In view of these results, a general picture emerges of how large proteins guide small ligands toward their active sites

The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection.

Bio-inspired noble-metal-free supramolecular systems were constructed by host-guest inclusions of an FeFe-hydrogenase mimic and an organic dye into cyclodextrins respectively, which give typically a ninefold increase in the TON, a sixteenfold enhancement in the quantum efficiency, and a threefold extension in lifetime as compared to the same system in the absence of cyclodextrin for photocatalytic H-2 production in aqueous solution., QC 20120903

The mannitol transporter EIImtl from Escherichia coli is responsible for the uptake of mannitol over the inner membrane and its concomitant phosphorylation. EIImtl is functional as a dimer and its membrane-embedded C domain, IICmtl, harbors one high affinity mannitol binding site. To characterize this domain in more detail the microenvironments of thirteen residue positions were explored by 5-fluorotryptophan (5-FTrp) fluorescence spectroscopy. Because of the simpler photophysics of 5-FTrp compared to Trp, one can distinguish between the two 5-FTrp probes present in dimeric IICmtl. At many labeled positions, the microenvironment of the 5-FTrps in the two protomers differs. Spectroscopic properties of three mutants labeled at positions 198. 251, and 260 show that two conserved motifs (Asn194-His195 and Gly254-Ile255-His256-Glu257) are located in well-structured parts of IICmtl. Mannitol binding has a large impact on the structure around position 198. while only minor changes are induced at positions 251 and 260. Phosphorylation of the cytoplasmic B domain of EIImtl is sensed by 5-FTrp at positions 30, 42, 251 and 260. We conclude that many parts of the IICmtl structure are involved in the sugar translocation. The structure of EIImtl, as investigated in this work. differs from the recently solved structure of a IIC protein transporting diacetylchitobiose, ChbC, and also belonging to the glucose superfamily of EII sugar transporters. In EIImtl, the sugar binding site is more close to the periplasmic face and the structure of the 2 protomers in the dimer is different, while both protomers in the ChbC dimer are essentially the same. (C) 2011 Elsevier B.V. All rights reserved.

Chiral beta-amino acids occur as constituents of various natural and synthetic compounds with potentially useful bioactivities. The pyridoxal 5'-phosphate (PLP)-dependent S-selective transaminase from Mesorhizobium sp. strain LUK (MesAT) is a fold type I aminotransferase that can be used for the preparation of enantiopure beta-Phe and derivatives thereof. Using x-ray crystallography, we solved structures of MesAT in complex with (S)-beta-Phe, (R)-3-amino-5-methylhexanoic acid, 2-oxoglutarate, and the inhibitor 2-aminooxyacetic acid, which allowed us to unveil the molecular basis of the amino acid specificity and enantioselectivity of this enzyme. The binding pocket of the side chain of a beta-amino acid is located on the 3'-oxygen side of the PLP cofactor. The same binding pocket is utilized by MesAT to bind the alpha-carboxylate group of an alpha-amino acid. A beta-amino acid thus binds in a reverse orientation in the active site of MesAT compared with an alpha-amino acid. Such a binding mode has not been reported before for any PLP-dependent aminotransferase and shows that the active site of MesAT has specifically evolved to accommodate both beta- and alpha-amino acids.

Reaction of Fe-3(CO)(12) with pyridinyl thioester ligand PyCH2SCOCH3 (L-1, Py = pyridin-2-yl) produced complex, [Fe-2(kappa-COCH3)(mu-SCH2Py)(CO)(5)] (1) (PyCH2S = pyridin-2-ylmethanethiolate). When complex 1 reacted with PPh3, a monosubstituted complex, [Fe-2(kappa-COCH3)(mu-SCH2Py)(CO)(4)PPh3] (2), was derived. Reaction of the same precursor with analogous thioester ligand PyCH2SCOPy (L-2) generated three novel diiron complexes, [Fe-2(kappa-Py)(mu-SCH2Py)(CO)(5)] (3), [Fe-2(kappa-Py)'(mu-SCH2Py)(CO)(5)] (4), and [Fe-2(kappa-Py)(mu-SCH2Py)(CO)(6)] (5). Complexes 3 and 4 are structural isomers. Complex 5 could be converted into complex 4 but the conversion from complex 5 to the isomer 3 was not observed. All the five complexes were fully characterised using FTIR, NMR, and other techniques. Their structures were determined using X-ray single crystal diffraction analysis. The oxidative formation of complexes 1, 3, 4, and 5 involved C-S and/ or C-C bonds cleavages. To probe possible mechanisms for these cleavages, DFT calculations were performed. From the calculations, viable reaction pathways leading to the formation of all the isolated products were delineated. The results of the theoretic calculations also allowed rationalisation of the experimental observations.

The diiron hydride [(mu-H)Fe-2(pdt)-(CO)(4)(dppv)](+) ([H-2](+), dppv = cis-1,2-C2H2(PPh2)(2)) is shown to be an effective photocatalyst for the H-2 evolution reaction (HER). These experiments establish the role of hydrides in photocatalysis by biomimetic diiron complexes. Trends in redox potentials suggests that other unsymmetrically substituted diiron hydrides are promising catalysts. Unlike previous catalysts for photo-HER, [H-2](+) functions without sensitizers: irradiation of [H-2] in the presence of triflic acid (HOTf) efficiently affords H-2. Instead of sacrificial electron donors, ferrocenes can be used as recyclable electron donors for the photocatalyzed HER, resulting in 4 turnovers.

The reactivity of [Fe-2(CO)(4)(kappa(2)-LL)(mu-pdt)](+) complexes (pdt = S(CH2)(3)S, propanedithiolate) with different substrates L' (L' = CO, MeCN, P(OMe)(3)) was investigated electrochemically in order to assess the influence of the chelating ligand kappa(2)-LL (LL = I-Me-CH2-I-Me (1(+)), dppe (2(+)); I-Me = 1-methylimidazol-2-ylidene). This latter ligand is effectively shown to affect the reactivity of the cations in different ways: when L' = CO, the adduct [Fe-2(CO)(4)(mu-CO)(kappa(2)-dppe)(mu-pdt)](+) (2-CO+) was clearly observed by cyclic voltammetry, whereas [Fe-2(CO)(4)(mu-CO)(kappa(2)-I-Me-CH2-I-Me)(mu-pdt)](+) (1-CO+) was not detected, although DFT calculations show that the energies of the products and the activation barriers to their formation are similar. When L' = MeCN, the adducts X-MeCN+ with X = 1, 2 are both observed, but the formation is easier when LL = dppe. Finally, the reaction of [Fe-2(CO)(4)(kappa(2)-I-Me-CH2-I-Me)(mu-pdt)](+) (1(+)) with P(OMe)(3) produces the disubstituted dication [Fe-2(CO)(2){P(OMe)3}(2)(kappa(2)-I-Me-CH2-I-Me)(mu-CO)(mu-pdt)](2+) (4(2+)) via a disproportionation reaction, while previous studies demonstrated that monocationic derivatives were obtained when LL = dppe. Complex 4[PF6](2) was fully characterized, and its X-ray crystal structure confirms the presence of a carbonyl ligand in a bridging position, which did not exist in the related P(OMe)(3)-substituted kappa(2)-dppe cations.

Key stereoelectronic properties of Desulfovibrio desulfuricans [FeFe]-hydrogenase (DdH) were investigated by quantum mechanical description of its complete inorganic core, which includes a Fe6S6 active site (the H-cluster), as well as two ancillary Fe4S4 assemblies (the F and F' clusters). The partially oxidized, active-ready form of DdH is able to efficiently bind dihydrogen, thus starting H2 oxidation catalysis. The calculations allow us to unambiguously assign a mixed Fe(II)Fe(I) state to the catalytic core of the activeready enzyme and show that H2 uptake exerts subtle, yet crucial influences on the redox properties of DdH. In fact, H2 binding can promote electron transfer from the H-cluster to the solvent-exposed F'-cluster, thanks to a 50% decrease of the energy gap between the HOMO (that is localized on the H-cluster) and the LUMO (which is centered on the F'-cluster). Our results also indicate that the binding of the redox partners of DdH in proximity of its F'-cluster can trigger one-electron oxidation of the H2-bound enzyme, a process that is expected to have an important role in H2 activation. Our findings are analyzed not only from a mechanistic perspective, but also in consideration of the physiological role of DdH. In fact, this enzyme is known to be able to catalyze both the oxidation and the evolution of H2, depending on the cellular metabolic requirements. Hints for the design of targeted mutations that could lead to the enhancement of the oxidizing properties of DdH are proposed and discussed. © 2011 American Chemical Society.

The effect of a (2,5)B boat conformation on xyloside reactivity has been investigated by studying the hydrolysis and glycosylation of a series of synthetic xyloside analogues based on a 2-oxabicyclo[2.2.2] octane framework, which forces the xylose analogue to adopt a (2,5)B conformation. The locked beta-xylosides were found to hydrolyze 100-1200 times faster than methyl beta-D-xylopyranoside, whereas the locked alpha-xylosides hydrolyzed up to 2 x 10(4) times faster than methyl alpha-D-xylopyranoside. A significant rate enhancement was also observed for the glycosylation reaction. The high reactivity of these conformers can be related to the imposition of a (2,5)B conformation, which ap-proximates a transition state (TS) boat conformation. In this way, the energy penalty required to go from the chair to the TS conformation is already paid. These results parallel and support the observation that the GH-11 xylanase family force their substrate to adopt a (2,5)B conformation to achieve highly efficient enzymatic glycosidic bond hydrolysis.

The formation of a dynamic hemithioacetal system and its application toward the discovery of ß-galactosidase inhibitors were successfully investigated. The designed dynamic system, which has a virtual character in neutral aqueous media, was subjected to a direct in situ identification of the best inhibitors by 1H STD NMR spectroscopy (ONPC : o-nitrophenyl-β-galactopyranoside)., QC 20100908

Hydrogen evolution was observed from the noble-metal-free catalyst systems, comprising Rose Bengal, BFx-bridged cobaloximes, and triethylamine, in an aqueous solution under irradiation of visible light. Two types of BFx-bridged cobaloximes namely, the annulated cobaloximes [Co(dmgBF(2))(2)(H2O)(2)] (1, dmgBF(2) = (difluoroboryl)dimethylglyoximate anion) and [Co(dpgBF(2))(2)(H2O)(2)] (2, dpgBF(2) =(difluoroboryl)diphenylglyoximate anion), and the clathrochelated cobaloximes [Co(dmg(BF)(2/3))(3)](BF4) (3) and [Co(dpg(BF)(2/3))(3)](BF4) (4)-were used as catalysts. Among the four cobalt complexes, complex 1 displayed the highest hydrogen-evolving efficiency, with turnovers up to 327. Complexes 2 and 4 that bear the diphenylglyoximate ligands exhibited much lower efficiencies as compared with their analogues 1 and 3 that have the dimethylglyoximate ligands. The hydrogen-evolving efficiency of the annulated cobalt(II) complex 1 that contains two labile axial ligands is more than three times as high as that of the encapsulated cobalt(III) complex 3 that has a single macrobicyclic ligand. The different pathways for formation of the cobalt(I) species from these two types of cobaloximes are discussed on the basis of the results obtained from fluorescence and laser flash photolysis spectroscopic studies., QC 20101130

The mechanism for H-2 cleavage in NiFe-hydrogenase has been reinvestigated with large models using both hybrid DFT by itself, or in a QM/MM scheme following the ONIOM approach. Heterolytic cleavage, with one hydrogen ending up as a bridging hydride and one as a proton on a cysteine ligand, was found to have a barrier slightly too high to be compatible with measured catalytic turnover rates. Alternative mechanisms were therefore investigated. In the finally suggested mechanism, heterolytic cleavage is used only as an initial step to generate a complex with nickel in oxidation state Ni(I). In the following cycles, H-2 is instead cleaved on nickel using an oxidative addition mechanism with a lower barrier. It was found that the ONIOM results for the reaction mechanism in NiFe-hydrogenase needed to be corrected by large model DFT results to be more reliable. This was mainly an effect of overestimation of polarization effects of the QM region by the MM region due to the particular treatment of the electrostatic interactions and the use of a standard (nonpolarizable) force field., authorCount :2

The exopolygalacturonase from Thermotoga maritima is the most thermoactive and thermostable pectinase known to date. Here we present its crystal structure at 2.05A resolution. High structural homology around the active site allowed us to propose a model for substrate binding, explaining the exo-cleavage activity and specificity for non-methylated saturated galacturonate at the non-reducing end. Furthermore, the structure reveals unique features that contribute to the formation of stable tetramers in solution. Such an oligomerization has not been observed before for polygalacturonases

Candida antarctica lipase B (CALB) is a promiscuous serine hydrolase that, besides its native function, catalyzes different side reactions, such as direct epoxidation. A single-point mutant of CALB demonstrated a direct epoxidation reaction mechanism for the epoxidation of alpha,beta-unsaturated aldehydes by hydrogen peroxide in aqueous and organic solution. Mutation of the catalytically active Ser105 to alanine made the previously assumed indirect epoxidation reaction mechanism impossible. Gibbs free energies, activation parameters, and substrate selectivities were determined both computationally and experimentally. The energetics and mechanism for the direct epoxidation in CALB Ser105Ala were investigated that the reaction proceeds through a two step-mechanism with formation of an oxyanionic intermediate. The active-site residue His224 functions as a general acid-base catalyst with support from Asp187. Oxyanion stabilization is facilitated by two hydrogen bonds from Thr40., QC20100608

The core structure (mu-SCH2)(2)NH[Fe-2(CO)(6)](5) of Fe-only hydrogenases active site model has been synthesized by the condensation of iron carbonyl sulfides, formaldehyde and silyl protected amine. Its monosubstituted complexes (mu-SCH2)(2)NH[Fe-2(CO)(5)PR3] (R = Ph (6), Me (7)) were accordingly prepared. The coordination configurations of 5 and 6 were characterized by X-ray crystallography. Protonation of complex 7 to form the N-protonated product occurs in an acetonitrile solution upon addition of triflic acid. The redox properties of these model complexes were studied by cyclic voltammetry., QC 20100525

Sulfurtransferases/rhodaneses (Str) are enzymes widely distributed in archaea, prokaryota and eukaryota, and catalyze the transfer of sulfur from a donor molecule to a thiophilic acceptor substrate. In this reaction, Str cycles between the sulfur-free and the sulfur-substituted form. Two-domain Str consist of two globular domains of nearly identical size and conformation connected by a short linker sequence, which is elongated in plant two-domain Str proteins compared to Str in other organisms. The two-domain Arabidopsis thaliana Str1 protein (At1g 79230) was expressed in Escherichia coli as a mature protein, as a variant without the elongated linker sequence, and as AtStr1C332S and AtStr1C339V. The persulfuration state of the purified recombinant proteins was investigated in the presence and absence of sulfur donors by fluorescence spectroscopy. The secondary structure was analyzed by circular dichroism (CD) in the far-UV range, while overall changes in tertiary structure were determined by CD in the near-UV range. Finally, protein stability was analyzed by tryptic digestion. The elongated linker sequence is essential for correct conformation and stability, and thereby affects the catalytic activity of AtStr1. Replacement of the catalytic cysteine residue C332 leads to higher rigidity of the molecule, whereas replacement of C339 does not lead to any conformational changes, providing evidence of the direct involvement of C339 in catalysis.

Comparative studies of the transglycosylation and hydrolytic activities have been performed on the Rhodothermus marinus beta-1,3-glucanase (laminarinase) and its M133A, M133C, and M133W mutants. The M133C mutant demonstrated near 20% greater rate of transglycosylation activity in comparison with the M133A and M133W mutants that was measured by NMR quantitation of nascent beta(1-4) and beta(1-6) linkages. To obtain kinetic probes for the wild-type enzyme and Met-133 mutants, p-nitrophenyl beta-laminarin oligosaccharides of degree of polymerisation 2-8 were synthesized enzymatically. Catalytic efficiency values, k (cat)/K (m), of the laminarinase catalysed hydrolysis of these oligosaccharides suggested possibility of four negative and at least three positive binding subsites in the active site. Comparison of action patterns of the wild-type and M133C mutant in the hydrolysis of the p-nitrophenyl-beta-D-oligosac- charides indicated that the increased transglycosylation activity of the M133C mutant did not result from altered subsite affinities. The stereospecificity of the transglycosylation reaction also was unchanged in all mutants; the major transglycosylation products in hydrolysis of p-nitrophenyl laminaribioside were beta-glucopyranosyl-beta-1,3-D-glucopy- ranosyl-beta-1,3-D-glucopyranose and beta-glucopyranosyl-beta-1, 3-D-glucopyranosyl-beta-1,3-D-glucpyranosyl-beta-1,3-D- glucopyranoxside., QC 20100525

A new biomimetic model of the active site of Fe-only hydrogenase [{(mu-SCH2)(2)N(2-C4H3O)} Fe-2(CO)(6)] (6) was synthesized and characterized by IR, H-1, C-13 NMR spectroscopy. The butterfly based core structure was also confirmed by X-ray diffraction. The electrochemistry data indicated that the reductive potential at -1.13 V versus Fc/Fc(+) by HClO4 is the lowest of that reported., QC 20100525

In 11 patients with a recessive congenital disorder, which we refer to as ¿the hypotonia-cystinuria syndrome,¿ microdeletion of part of the SLC3A1 and PREPL genes on chromosome 2p21 was found. Patients present with generalized hypotonia at birth, nephrolithiasis, growth hormone deficiency, minor facial dysmorphism, and failure to thrive, followed by hyperphagia and rapid weight gain in late childhood. Since loss-of-function mutations in SLC3A1 are known to cause isolated cystinuria type I, and since the expression of the flanking genes, C2orf34 and PPM1B, was normal, the extended phenotype can be attributed to the deletion of PREPL. PREPL is localized in the cytosol and shows homology with prolyl endopeptidase and oligopeptidase B. Substitution of the predicted catalytic residues (Ser470, Asp556, and His601) by alanines resulted in loss of reactivity with a serine hydrolase-specific probe. In sharp contrast to prolyl oligopeptidase and oligopeptidase B, which require both aminoterminal and carboxyterminal sequences for activity, PREPL activity appears to depend only on the carboxyterminal domain. Taken together, these results suggest that PREPL is a novel oligopeptidase, with unique structural and functional characteristics, involved in hypotonia-cystinuria syndrome.

Multiple non-active site interactions between ribonuclease A (RNAse) and selected target molecules were investigated using nano-electrospray ionization mass spectrometry (nano-ESI-MS). Among the building blocks of RNA, phosphate and ribose showed such multiple interactions. Multiple phosphate interactions survived a high cone voltage, while multiple interactions with D-ribose disappeared already at a low cone voltage. Using nano-ESI-MS, only cytosine among the individual bases appeared to interact with RNAse. Interestingly, guanosine binds to the RNAse surface at high cone voltage, probably as a result of cooperative binding of the sugar and the guanine base. Upon binding of deoxycytidine oligonucleotides with six (dC(6)), nine (dC(9)) and twelve (dC(12)) deoxycytidine nucleotide units to RNAse, the dC(12) Unit showed the strongest interaction. Upon collision-induced dissociation (CID) of the RNAse/dC(6) complex, this complex survived dissociation at an energy level where covalently bound cytosine from dC(6) was lost. This is in contrast to CID of RNAse complexed with mononucleotide cytidine 2'-monophosphate (CMP), which dissociates from the protein without breaking of covalent bonds., QC 20100913

The metal- sulphur active sites of hydrogenases catalyse hydrogen evolution or uptake at rapid rates. Understanding the structure and function of these active sites - through mechanistic studies of hydrogenases(1 - 4), synthetic assemblies(5 - 12) and in silico models(13 - 15) - will help guide the design of new materials for hydrogen production or uptake(16). Here we report the assembly of the iron- sulphur framework of the active site of iron- only hydrogenase ( the H- cluster), and show that it functions as an electrocatalyst for proton reduction. Through linking of a di- iron subsite to a {4Fe4S} cluster, we achieve the first synthesis of a metallosulphur cluster core involved in small- molecule catalysis. In addition to advancing our understanding of the natural biological system, the availability of an active, free- standing analogue of the H- cluster may enable us to develop useful electrocatalytic materials for application in, for example, reversible hydrogen fuel cells. ( Platinum is currently the preferred electrocatalyst for such applications, but is expensive, limited in availability and, in the long term, unsustainable(17).)

Heterologous proteins secreted by yeast and fungal expression hosts are occasionally degraded at basic amino acids. We cloned Pichia pastoris homologs of the Saccharomyces cerevisiae basic residue-specific endoproteases Kex2 and Yps1 to evaluate their involvement in the degradation of a secreted mammalian gelatin. Disruption of the P. pastoris KEX2 gene prevented proteolysis of the foreign protein at specific monoarginylic sites. The S. cerevisiae -factor preproleader used to direct high-level gelatin secretion was correctly processed at its dibasic site in the absence of the prototypical proprotein convertase Kex2. Disruption of the YPS1 gene had no effect on gelatin degradation or processing of the -factor propeptide. When both the KEX2 and YPS1 genes were disrupted, correct precursor maturation no longer occurred. The different substrate specificities of both proteases and their mutual redundancy for propeptide processing indicate that P. pastoris kex2 and yps1 single-gene disruptants can be used for the -factor leader-directed secretion of heterologous proteins otherwise degraded at basic residues.

The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations. The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K-M and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3) + Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop., QC 20100525

Two carboxylic acid side chains can, depending on their geometry and environment, share a proton in a hydrogen bond and form a carboxyl-carboxylate pair. In the Trichoderma reesei cellobiohydrolase Cel6A structure, five carboxyl-carboxylate pairs are observed. One of these pairs (D175-D221) is involved in catalysis, and three other pairs are found in, or close to the two surface loops covering the active site tunnel of the catalytic domain. To stabilize Cel6A at alkaline pH values, where deprotonation of the carboxylic acids leads to repulsion of their side chains, we designed two mutant enzymes. In the first mutant, one carboxyl-carboxylate pair (E107-E399) was replaced by a corresponding amide-carboxylate pair (Q107-E399), and in the second mutant, all three carboxyl-carboxylate pairs (E107-E399, D170-E184, and D366-D419) were mutated in a similar manner. The unfolding studies using both intrinsic tryptophan fluorescence and far-ultraviolet circular dichroism spectroscopy at different pH values demonstrate that the unfolding temperature (T-m) of both mutants has changed, resulting in destabilization of the mutant enzymes at acidic pH and stabilization at alkaline pH. The effect of stabilization seems additive, as a Cel6A triple mutant is the most stable enzyme variant. This increased stability is also reflected in the 2- or 4-fold increased half-life of the two mutants at alkaline pH, while the catalytic rate on cellotetraose (at t = 0) has not changed. Increased operational stability at alkaline pH was also observed on insoluble cellulosic substrates. Local conformational changes are suggested to take place in the active site loops of Cel6A wild-type enzyme at elevated pHs (pH 7), affecting to the end-product spectrum on insoluble cellulose. The triple mutant does not show such pH-dependent behavior. Overall, our results demonstrate that carboxyl-carboxylate pair engineering is a useful tool to alter pH-dependent protein behavior., QC 20100525

The effects of a variety of ortho-substituents (CH3, OH, OCH3, SH, SCH3, NH2, NO2, F, Cl, CN, and imidazole) on the acidity of phenol are investigated using hybrid density functional theory. Substitutions are made at the ortho-position to model modified tyrosine residues found in enzymes. Although the experimental trends are reproduced, the calculations tend to exaggerate the substituent effects. It is shown that the cysteine cross-link to tyrosine, present in the radical enzyme galactose oxidase, has a small effect on the pK(a) of the residue. The histidine cross-link present in cytochrome c oxidase, on the other hand, will contribute more to. lower the pKa. Comparing the substituent effects on the O-H bond strengths and the acidities, no simple correlation is found between the two., QC 20100525

We have found chlorine kinetic isotope effects on the dehalogenation catalyzed by haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 to be 1.0045 ± 0.0004 for 1,2-dichloroethane and 1.0066 ± 0.0004 for 1-chlorobutane. The latter isotope effect approaches the intrinsic chlorine kinetic isotope effect for the dehalogenation step. The intrinsic isotope effect has been modeled using semiempirical and DFT theory levels using the ONIOM QM/QM scheme. Our results indicate that the dehalogenation step is reversible; the overall irreversibility of the enzyme-catalyzed reaction is brought about by a step following the dehalogenation., Addresses: Paneth P, Lodz Tech Univ, Dept Chem, Zeromskiego 116, PL-90924 Lodz, Poland. Lodz Tech Univ, Dept Chem, PL-90924 Lodz, Poland. Univ Uppsala, Ctr Biomed, Dept Biochem, S-75123 Uppsala, Sweden. Univ Uppsala, Dept Chem, S-75121 Uppsala, Sweden.

Density functional methods, alone and together with molecular mechanics, are used to study the catalytic mechanism of galactose oxidase. This enzyme catalyzes the conversion of primary alcohols to the corresponding aldehydes, coupled with reduction of dioxygen to hydrogen peroxide. It is shown that the proposed mechanism for this enzyme is energetically feasible. In particular the barrier for the postulated rate-limiting hydrogen atom, transfer between the substrate and the tyrosyl radical, located at equatorial Tyr272, is very plausible. We propose that the radical site, prior to the initial proton transfer step, is located at the axial tyrosine (Tyr495). The radical is transferred to the equatorial tyrosine (Tyr272) simultaneously with the proton transfer. It is, furthermore, argued that the electron transfer from the ketyl radical intermediate to Cu(II) cannot be very exothermic, because this would render the oxygen reduction steps rate-limiting. Finally, the cysteine cross-link on the active site tyrosine is shown to have very minor effects on the energetics of the reaction., QC 20100525

In recent experimental studies on the E441Q mutant of ribonucleotide reductase, a new substrate radical was discovered on the minute time scale. This radical is kinetically coupled to a cysteine-based radical appearing on the 10 s time scale. In the present study, density functional calculations have been performed to investigate the nature of these radicals. The most interesting result is that a very stable substrate radical was found, which lies outside the normal substrate pathway. This radical is so stable that its creation has to be avoided by the enzyme, or the substrate reactions would be slowed by several orders of magnitude. It is suggested that the enzyme accomplishes this task by considerably straining the mobility of the Cys225 residue. A previously suggested reaction mechanism is modified to take these recent findings into account. The modification does not significantly change the energetics of the model reactions., QC 20100525

Combining sequence analysis, structure prediction, and site-directed mutagenesis, we have investigated the mechanism of catalysis and substrate binding by the apoptotic mitochondrial nuclease EndoG, which belongs to the large family of DNA/RNA non-specific ββα-Me-finger nucleases. Catalysis of phosphodiester bond cleavage involves several highly conserved amino acid residues, namely His143, Asn174, and Glu182 required for water activation and metal ion binding, as well as Arg141 required for proper substrate binding and positioning, respectively. These results indicate that EndoG basically follows a similar mechanism as the Serratia nuclease, the best studied representative of the family of DNA/RNA non-specific nucleases, but that differences are observed for transition state stabilisation. In addition, we have identified two putative DNA/RNA binding residues of bovine EndoG, Arg135 and Arg186, strictly conserved only among mammalian members of the nuclease family, suggesting a similar mode of binding to single and double-stranded nucleic acid substrates by these enzymes. Finally, we demonstrate by ectopic expression of active and inactive variants of bovine EndoG in HeLa and CV1-cells that extramitochondrial active EndoG by itself induces cell death, whereas expression of an enzymatically inactive variant does not. © 2004 Elsevier Ltd. All rights reserved.

Combining sequence analysis, structure prediction, and site-directed mutagenesis, we have investigated the mechanism of catalysis and substrate binding by the apoptotic mitochondrial nuclease EndoG, which belongs to the large family of DNA/RNA non-specific ββα-Me-finger nucleases. Catalysis of phosphodiester bond cleavage involves several highly conserved amino acid residues, namely His143, Asn174, and Glu182 required for water activation and metal ion binding, as well as Arg141 required for proper substrate binding and positioning, respectively. These results indicate that EndoG basically follows a similar mechanism as the Serratia nuclease, the best studied representative of the family of DNA/RNA non-specific nucleases, but that differences are observed for transition state stabilisation. In addition, we have identified two putative DNA/RNA binding residues of bovine EndoG, Arg135 and Arg186, strictly conserved only among mammalian members of the nuclease family, suggesting a similar mode of binding to single and double-stranded nucleic acid substrates by these enzymes. Finally, we demonstrate by ectopic expression of active and inactive variants of bovine EndoG in HeLa and CV1-cells that extramitochondrial active EndoG by itself induces cell death, whereas expression of an enzymatically inactive variant does not. © 2004 Elsevier Ltd. All rights reserved.