Search

[FeFe]-Hydrogenases harbor a {2Fe3S} assembly bearing two CO and two CN- groups, a mu-CO ligand, and a vacant coordination site trans to the mu-CO group. Recent theoretical results obtained studying the isolated {2Fe3S} subsite indicated that one of the CN- ligands can easily move from the crystallographic position to the coordination site trans to the mu-CO group; such an isomerization would have a major impact on substrates and inhibitors binding regiochemistry and, consequently, on the catalytic mechanism. To shed light on this crucial issue, we have carried out hybrid QM/MM and free energy perturbation calculations on the whole enzyme, which demonstrate that the protein environment plays a crucial role and maintains the CN- group fixed in the position observed in the crystal structure; these results strongly support the hypothesis that the vacant coordination site trans to the mu-CO group has a crucial functional relevance both in the context of CO-mediated inhibition of the enzyme and in dihydrogen oxidation/evolution catalysis.

Adaptation to both high and low temperatures requires proteins with special properties. While organisms living at or close to the boiling point of water need to have proteins with increased stability, other properties are required at temperatures close to the freezing point of water. Indeed, it has been shown that enzymes adapted to cold environments are less resistant to heat with a concomitant increased activity as compared to their warm-active counter-parts. Several recent studies have pointed in the direction that electrostatic interactions play a central role in temperature adaptation, and in this study we investigate the role such interactions have in adaptation of elastase from Atlantic salmon and pig. Molecular dynamics (MD) simulations have been used to generate structural ensembles at 283 and 310 K of the psychrophilic and mesophilic elastase, and a total of eight 12 ns simulations have been carried out. Even though the two homologues have a highly similar three-dimensional structure, the location and number of charged amino acids are very different. Based on the simulated structures we find that very few salt-bridges are stable throughout the simulations, and provide little stabilization/destabilization of the proteins as judged by continuum electrostatic calculations. However, the mesophilic elastase is characterized by a greater number of salt-bridges as well as a putative salt-bridge network close to the catalytic site, indicating a higher rigidity of the components involved in the catalytic cycle. In addition, subtle differences are also found in the electrostatic potentials in the vicinity of the catalytic residues, which may explain the increased catalytic efficiency of the cold-adapted elastase. © 2006 Elsevier Inc. All rights reserved.

The singlet and triplet state potential energy surfaces (PES) of Fe-2(CO)(8) are explored by means of density functional theory (DFT) techniques. The two PES have different global mimima: the dibriged C-2v isomer for the singlet and the unbridged D-2d isomer for the triplet. The sign of the energy gap between singlet and triplet global minima depends on the type of adopted DFT functional: hybrid functionals predict the triplet is more stable than the singlet, but the opposite applies to generalized gradient approximated (GGA) functionals. The analysis of the computed CO stretching frequencies demonstrates that the experimental data for the unbridged form is compatible also with the unbridged triplet D-2d isomer. Starting from these two facts, the electronic structure of unbridged D-2d Fe-2(CO)(8) is discussed herein. Single-point energy computations at the coupled-cluster single and double (CCSD) level favor the D-2d triplet state.

Fe-only hydrogenases are enzymes that catalyze dihydrogen production or oxidation, due to the presence of an unusual Fe6S6 cluster (the so-called H-cluster) in their active site, which is composed of a Fe2S2 subsite, directly involved in catalysis, and a classical Fe4S4 cubane cluster. Here, we present a hybrid quantum mechanical and molecular mechanical (QM/MM) investigation of the Fe-only hydrogenase from Desulfovibrio desulfuricans, in order to unravel key issues regarding the activation of the enzyme from its completely oxidized inactive state (H-ox(inac)) and the influence of the protein environment on the structural and catalytic properties of the H-cluster. Our results show that the Fe2S2 subcluster in the (FeFeII)-Fe-II redox statewhich is experimentally observed for the completely oxidized form of the enzymebinds a water molecule to one of its metal centers. The computed QM/MM energy values for water binding to the diferrous subsite are in fact over 70 kJ mol(-1); however, the affinity toward water decreases by 1 order of magnitude after a one-electron reduction of H-ox(inact), thus leading to the release of coordinated water from the H-cluster. The investigation of a catalytic cycle of the Fe-only hydrogenase that implies formation of a terminal hydride ion and a di(thiomethyl)amine (DTMA) molecule acting as an acid/base catalyst indicates that all steps have reasonable reaction energies and that the influence of the protein on the thermodynamic profile of H-2 production catalysis is not negligible. QM/MM results show that the interactions between the Fe2S2 subsite and the protein environment could give place to structural rearrangements of the H-cluster functional for catalysis, provided that the bidentate ligand that bridges the iron atoms in the binuclear subsite is actually a DTMA residue.

The new complexes Fe-2(S(2)CnH(2)n)(CO)(2)(dppv)(2) (n = 2, 3; dppv = cis-1,2-C2H2(PPh2)(2)) form adducts with AlBr3 and B(C6F5)(3), which adopt the "rotated structure" proposed for the active site of the Fe-only hydrogenases - the propanedithiolate is significantly more Lewis basic due to nonbonded interactions between the dithiolate strap and the ligands on Fe.

The complex (S2C3H6)[Fe-2(CO)(5)P(NC4H8)(3)] (A), a recently synthesized functional model of the active site of Fe-hydrogenases, is able to electrocatalyze proton reduction, leading to molecular hydrogen evolution. Experimental results suggested that the presence of the electron donor P(NC4H8)(3) ligand in A could favor the formation of a mu-CO species similar to that observed in the enzymatic cluster. However, insight into the structural features of key catalytic intermediates deriving from reduction and protonation of A was still lacking. Here we present results obtained using density functional theory to evaluate structures, relative stabilities, and spectroscopic properties of several species relevant for the electrocatalytic H-2 evolving process. The results enabled us to unravel the structure of the mu-CO complex experimentally detected after monoelectronic reduction of A. Moreover, we show that the introduction of the large electron-donor ligand P(NC4H8)(3) in the biomimetic complex does not favour the stabilization of terminal-hydrido adducts, which are expected to be very reactive in terms of H-2 production. The comparison of our findings with previous theoretical and experimental results obtained on similar model complexes suggests that the introduction of an electron donor ligand as good as P(NC4H8)(3), but less sterically demanding, could represent a better choice to facilitate the formation of mu-CO complexes more closely resembling the structure of the enzymatic cluster. ((C) Wiley-VCH Verlaq GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007).

Reaction of R-styreneoxide with glycine-tert-butylester yielded amino alcohols of the general formula (NRRR3)-R-1-R-2, where R-1 = CH2COO t Bu and R-2 = R-3 = 2-phenyl-2-hydroxyethyl (H(2)LA); R-2 = 2-phenyl-2-hydroxyethyl and R-3 = 1-phenyl-2-hydroxyethyl (H2LB); R-2 = H and R-3 = 2-phenyl-2-hydroxyethyl (HLC); and R-2 = H and R-3 = 1-phenyl-2-hydroxyethyl (HLD). The corresponding reaction with sarcosine-tert-butylester and subsequent hydrolysis provided the zwitterion (CH3){CH2CHPh(OH)}(CH2CO2-), HLE* (asterisk refers to unprotected carboxylate). Reaction of these ligands with VO(OiPr)(3) in CH2Cl2 gave the oxovanadium(V) complexes [VOL(OiPr)(2)] and [VOL2-(OiPr)] (for L-C and L-D) or, when reacted in the presence of MeOH, [VOL'(OMe)], where L' represents the methyl ester of L-A, L-B, and L-E. The crystal and molecular structures of R-HLC, S-HLD, R,S-HLE*center dot H2O, and Lambda[VO(R,S-L-B')OMe] have been determined. The complex [VOLB'(OMe)] contains vanadium in a distorted trigonal-bipyramidal array (tau = 0.72), the oxo group in the equatorial plane, and methoxide and N in the apical positions, and thus, it structurally models the active center of vanadate-dependent haloperoxidases. The structure and the bonding parameters, including a particularly long d(V-N) of 2.562 angstrom, are backed up by DFT calculations. The isolated oxovanadium(V) complexes and the in situ systems L VO(OiPr)(3) catalyze the oxidation, by cumylhydroperoxide HO2R', of prochiral sulfides (MeSPh, MeSp-Tol, PhSBn) to chiral sulfoxides plus some sulfone. The best results with respect to enantioselectivity (enantiomeric excess (ee) = 38%) were obtained with the system VO(OiPr)(3)/L-A, and the best selectivity with respect to sulfoxide (100%) was obtained with [VOLA(OiPr)]. The reaction with the hexacoordinated [VO(OMe)(HOMe)L-D*] was very slow. Oxidation of PhSBn is faster than that of MeSPh and MeSpTol. Turn-over numbers are up to 60 mol of sulfoxide mol(-1) of catalyst h(-1) (-20 degrees

The di-iron complex Fe-2(S2C3H6)(CO)(6) (a), one of the simplest functional models of the Fe-hydrogenases active site, is able to electrocatalyze proton reduction. In the present study, the H-2 evolving path catalyzed by a has been characterized using density functional theory. It is showed that, in the early stages of the catalytic cycle, a neutral mu-H adduct is formed; monoelectron reduction and subsequent protonation can give rise to a diprotonated neutral species (a-mu H-SH), which is characterized by a mu-H group, a protonated sulfur atom, and a CO group bridging the two iron centers, in agreement with experimental IR data indicating the formation of a long-lived mu-CO species. H-2 release from a-mu H-SH, and its less stable isomer a-H-2 is kinetically unfavorable, while the corresponding monoanionic compounds (a-mu H-SH- and a-H-2(-)) are more reactive in terms of dihydrogen evolution, in agreement with experimental data. The key species involved in electrocatalysis have structural features different from the hypothetical intermediates recently proposed to be involved in the enzymatic process, an observation that is possibly correlated with the reduced catalytic efficiency of the biomimetic di-iron assembly.

Density functional theory has been used to investigate the structural, electronic, and reactivity properties of an established functional model for vanadium-dependent haloperoxidases, K[VO(O-2)Hheida] (Hheida(2-) = 2,2'-[(2-hydroxyethyl)imino]diacetate). Possible solution species were determined on the basis of potential exogenous donors present under the conditions necessary for reactivity. The energetically favored solution-state species is a 1:1 complex of Hheida and vanadium with a coordinated hydroxyethyl donor trans to the vanadium-oxido bond which is in agreement with the reported solid-state structure for K[VO(O-2)Hheida]. Transition states of the oxidation reaction were located for four substrates: chloride, bromide, iodide, and dimethyl sulfide. The role of protonation and its effects on reactivity were examined for each substrate. Protonation of the peroxido moiety leads to a significant drop in the activation barrier for oxidation, In contrast no transition states could be located for an oxido-transfer process involving the oxido ligand. Barriers of activation calculated for halide oxidation were similar, providing support to the hypothesis that the pK(a), of the halide in acetonitrile is responsible for the decrease in reactivity between I-, Br-, and Cl-. The results presented herein provide a mechanistic correlation between a functional model and the enzyme, making K[VO(02)Hheida] a "complete" functional model for vanadium-dependent haloperoxidase. ((c) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Polynucleotide phosphorylase (PNPase) is a phosphate-dependent 3' to 5' exonuclease widely diffused among bacteria and eukaryotes. The enzyme, a homotrimer, can also be found associated with the endonuclease RNase E and other proteins in a heteromultimeric complex, the RNA degradosome. PNPase negatively controls its own gene (pnp) expression by destabilizing pnp mRNA. A current model of autoregulation maintains that PNPase and a short duplex at the 5'-end of pnp mRNA are the only determinants of mRNA stability. During the cold acclimation phase autoregulation is transiently relieved and cellular pnp mRNA abundance increases significantly. Although PNPase has been extensively studied and widely employed in molecular biology for about 50 years, several aspects of structure-function relationships of such a complex protein are still elusive. In this work, we performed a systematic PCR mutagenesis of discrete pnp regions and screened the mutants for diverse phenotypic traits affected by PNPase. Overall our results support previous proposals that both first and second core domains are involved in the catalysis of the phosphorolytic reaction, and that both phosphorolytic activity and RNA binding are required for autogenous regulation and growth in the cold, and give new insights on PNPase structure-function relationships by implicating the alpha-helical domain in PNPase enzymatic activity. (c) 2006 Elsevier Masson SAS. All rights reserved.

Phosphatidylinositol 3-kinase (PI3K) and serine/threonine protein kinase B (PKB or Akt) pathways regulate important cellular processes and are related to a number of human pathologies, such as cancer. The development of kinase inhibitors, with particular attention to small molecule analogues of natural phosphoinositides for pathway interuption and therapeutic applications will be reviewed. © 2006 Bentham Science Publishers Ltd.

Background: Metabolic pathway manipulation for improving the properties and the productivity of microorganisms is becoming a well established concept. For the production of important metabolites, but also for a better understanding of the fundamentals of cell biology, detailed studies are required. In this work we analysed the lactate production from metabolic engineered Succharomyces cerevisiae cells expressing a heterologous lactate clehydrogenase (LDH) gene. The LDH gene expression in a budding yeast cell introduces a novel and alternative pathway for the NAD+ regeneration, allowing a direct reduction of the intracellular pyruvate to lactate, leading to a simultaneous accumulation of lactate and ethanol. Results: Four different S. cerevisiae strains were transformed with six different wild type and one mutagenised LDH genes, in combination or not with the over-expression of a lactate transporter. The resulting yield values (grams of lactate produced per grams of glucose consumed) varied from as low as 0,0008 to as high as 0.52 g g-1. In this respect, and to the best of our knowledge, higher redirections of the glycolysis flux have never been obtained before without any disruption and/or limitation of the competing biochemical pathways. Conclusion: In the present work it is shown that the redirection of the pathway towards the lactate production can be strongly modulated by the genetic background of the host cell, by the source of the heterologous Ldh enzyme, by improving its biochemical properties as well as by modulating the export of lactate in the culture media. © 2006 Branduardi et al; licensee Biomed Central Ltd.

The valence electronic excited states of Fe2(CO)9 have been studied using the time-dependent density functional theory (TDDFT). Both tribridged D3h and monobridged C2V structures have been considered, and the structure of selected low-lying singlet and triplet excited states have been optimized on the basis of the TDDFT analytical gradient. Optimized excited-state geometries are used to obtain an insight into certain aspects of the Fe2- (CO)9 photochemistry. The Fe2(CO)9 (D3h) first triplet and second singlet excited states are unbound with respect to dibridged Fe2(CO)8 + CO, and the first two monobridged Fe2(CO)9 (C2V) singlet states are unbound with respect to the Fe(CO)5 + Fe(CO)4 dissociation. These results are discussed in light of the experimental data available

Density functional theory (DFT) has been used to investigate the catalytic properties of the isolated vanadium cofactor found in vanadium haloperoxidases, with a particular emphasis on the steps going from the resting form of the cofactor to the peroxo complex. Computation of transition states, intermediate species, and UV-vis spectra, as well as comparison of reaction energies, demonstrated the important role of protonation in cofactor activation. This illustrates that the resting form of the vanadium cofactor reacts with hydrogen peroxide according to a mechanism that implies formation of an aqua complex, release of the apical water molecule according to a dissociative pathway, and binding of hydrogen peroxide to vanadium. This process leads to a side-on peroxo species corresponding to the peroxo form observed in the enzyme. In addition, it appears that an acid-base catalysts strongly accelerates the conversion to the side-on peroxo form. The comparison of computed and experimental UV-vis spectra corroborated the proposed reaction pathway and allowed us to explain the effects of the vanadium ligands on the electronic properties of the cofactor. © 2006 American Chemical Society.

Molecular dynamics simulations of representative mesophilic and psycrophilic elastases have been carried out at different temperatures to explore the molecular basis of cold adaptation inside a specific enzymatic family. The molecular dynamics trajectories have been compared and analyzed in terms of secondary structure, molecular flexibility, intramolecular and protein-solvent interactions, unravelling molecular features relevant to rationalize the efficient catalytic activity of psychrophilic elastases at low temperature. The comparative molecular dynamics investigation reveals that modulation of the number of protein-solvent interactions is not the evolutionary strategy followed by the psycrophilic elastase to enhance catalytic activity at low temperature. In addition, flexibility and solvent accessibility of the residues forming the catalytic triad and the specificity pocket are comparable in the cold- and warm-adapted enzymes. Instead, loop regions with different amino acid composition in the two enzymes, and clustered around the active site or the specificity pocket, are characterized by enhanced flexibility in the cold-adapted enzyme. Remarkably, the psycrophilic elastase is characterized by reduced flexibility, when compared to the mesophilic counterpart, in some scattered regions distant from the functional sites, in agreement with hypothesis suggesting that local rigidity in regions far from functional sites can be beneficial for the catalytic activity of psychrophilic enzymes. (c) 2006 Elsevier B.V. All rights reserved.

The three-dimensional (3D) structure of the catalytic domain of Gas1p, a protein belonging to the only family of beta-(1,3)-glucan transferases so far identified in yeasts and some pathogenic fungi (family GH-72), has been predicted by combining results derived from threading methods, multiple sequence alignments and secondary-structure predictions. The 3D model has allowed the identification of several residues that are predicted to play a crucial role in structural integrity, substrate recognition and catalysis. In particular, the model of the catalytic domain can be useful for designing site-directed mutagenesis experiments and for developing inhibitors of Gas1p enzymatic activity.

Density functional theory (DFT) has been recently used to explore the chemistry of models of the active site of [NiFe] and [Fe] hydrogenases, which are enzymes that catalyse the reversible oxidation of H2. Results from recent theoretical investigations aimed at characterizing relevant intermediate species in the catalytic cycle of hydrogenases, clarifying the structural and electronic properties of the bimetallic cofactors, as well as the key factors responsible for H2 activation, are reviewed. The role of theoretical contributions in the investigation of synthetic models related to the enzymatic metal cofactors is also discussed, showing how 'in silico' coordination chemistry can nicely complement experimental studies in the characterization of relevant species. © 2004 Elsevier B.V. All rights reserved.

Density functional theory has been used to investigate the reaction between H-2 and [Ni(NHPnPr(3))('S3')] or [Pd(NHPnPr(3))('S3')], where 'S3' = bis(2-sulfanylphenyl)sulfide(2-), which are among the few synthetic complexes featuring a metal coordination environment similar to that observed in the [NiFe] hydrogenase active site and capable of catalyzing H-2 heterolytic cleavage. Results allowed us to unravel the reaction mechanism, which is consistent with an oxidative addition-hydrogen migration pathway for [Ni(NHPnPr(3))('S3')], whereas metathesis is also possible with [Pd(NHPnPr(3))('S3')]. Unexpectedly, H-2 binding and activation implies structural reorganization of the metal coordination environment. It turns out that the structural rearrangement in [Ni(NHPnPr(3))('S3')] and [Pd(NHPnPr(3))('S3')] can take place due to the peculiar structural features of the Ni and Pd ligands, explaining the remarkable catalytic properties. However, the structural reorganization is the most unfavorable step along the H-2 cleavage pathway (Delta G > 100 kJ mol(-1)), an observation that is relevant for the design and synthesis of novel biomimetic catalysts.

Cdc25Mm is a mammalian Ras-specific guanine nucleotide exchange factor (GEF). By homology modeling we show that it shares with Sos-GEF the structure of the putative catalytic HI hairpin where the dominant negative T1184E mutation is located. Similarly to Cdc25MmT1184E, the isolated wild-type and mutant hairpins retain the ability to displace Ras-bound nucleotide, originate a stable Ras/GEF complex and downregulate the Ras pathway in vivo. These results indicate that nucleotide re-entry and Ras/GEF dissociation--final steps in the GEF catalytic cycle--require GEF regions different from the HI hairpin. GEF down-sizing could lead to development of novel Ras inhibitors.

We have previously demonstrated that the cyclin-dependent kinase inhibitor (Cki) Sic1 of Saccharomyces cerevisiae is phosphorylated in vitro by the CK2 kinase on Ser(201) residue. Moreover, we have collected evidence showing that Sic1 is functionally and structurally related to mammalian Cki p27(Kip1) and binds to the mammalian Cdk2/cyclin A complex with a similar mode of inhibition. In this paper, we use SPR analysis to investigate the binding of Sic1 to the catatytic and regulatory subunits of CK2. Evidence is presented showing that phosphorylation of Sic1 at the CK2 consensus site QES(201)EDEED increases the binding of a Sic1-derived peptide to the Cdk2/cyclin A complex, a functional homologue of the yeast Cdk1/Clb5,6. Moreover, Sic1 fully phosphorylated in vitro on Ser(201) by CK2 is shown to be a stronger inhibitor of the Cdk/cyclin complexes than the unphosphorylated protein. Taken together, these data disclose the possibility that CK2 plays a role in the regulation of Sic1 activity.

The double-histone fold is a rare protein fold in which two consecutive regions characterized by the typical structure of histones assemble together, thus giving a histone pseudodimer. Previously, this fold was found in a few prokaryotic histones and in the regulatory region of guanine-nucleotide exchange factors of the Sos family. Standard methods of sequence comparison did not allow us to find new proteins containing a histone pseudodimer, as previously reported (Sondermann et al. 2003). However, a deeper investigation of protein sequences showed that the two histone folds included in Sos proteins share significant sequence similarity with nucleosomal histones. On the basis of this observation, we applied a specific strategy of sequence-homology search, which led to the identification of a new group of histone pseudodimers in Cca3 and proteins similar to Cca3 (Cca3S). A homology model of the histone pseudodimer included in rat Cca3 was constructed. A subsequent structure-function relationship study revealed that the histone pseudodimers included in Cca3 and Cca3S proteins, but not those present in Sos proteins, could retain the ability of mediating protein-DNA interactions, and could consequently act as DNA-binding modules. © Springer-Verlag 2005.

The calcium ion was proposed to be involved in protein structure stabilization against thermal and proteolytic degradation, such as autolysis phenomena, in trypsin-like serine proteases. However, molecular details related to the role played by the metal ion are still largely unknown. Several molecular dynamics simulations of 6 ns have been used to investigate the dynamic behavior of bovine and salmon trypsins in calcium-bound and calcium-free forms, with the aim of evaluating the role of the calcium ion in trypsin three-dimensional structure and autoproteolysis propensity. It turned out that the calcium-free trypsins are characterized by a more flexible structure, revealing structure-function relationships connecting Ca2+ binding and autoproteolysis propensity. In particular, the removal of Ca2+ not only increases the flexibility of regions around its binding site, in the N-terminal domain, but also leads to channeling of the fluctuations to remote sites in the C-terminal domain, possibly involving the interdomain loop. Two primary autolysis sites are strongly influenced by calcium binding (R117 and K188) in bovine trypsin, whereas Ca2+ plays a less crucial role in salmon trypsin. © 2005 American Chemical Society.

Background: Histones are short proteins involved in chromatin packaging; in eukaryotes, two H2a-H2b and H3-H4 histone dimers form the nucleosomal core, which acts as the fundamental DNA-packaging element. The double histone fold is a rare globular protein fold in which two consecutive regions characterized by the typical structure of histones assemble together, thus originating a histone pseudodimer. This fold is included in a few prokaryotic histones and in the regulatory region of guanine nucleotide exchange factors of the Sos family. For the prokaryotic histones, there is no direct structural counterpart in the nucleosomal core particle, while the pseudodimer from Sos proteins is very similar to the dimer formed by histones H2a and H2b Results: The absence of a H3-H4-like histone pseudodimer in the available structural databases prompted us to search for proteins that could assume such fold. The application of several secondary structure prediction and fold recognition methods allowed to show that the viral protein gi 22788712 is compatible with the structure of a H3-H4-like histone pseudodimer. Further in silico analyses revealed that this protein module could retain the ability of mediating protein-DNA interactions, and could consequently act as a DNA-binding domain. Conclusion: Our results suggest a possible functional role in viral pathogenicity for this novel double histone fold domain; thus, the computational analyses here reported will be helpful in directing future biochemical studies on gi 22788712 protein.

In Saccharomyces cerevisiae, Sic1, an inhibitor of Cdk (cyclin-dependent kinase), blocks the activity of S-Cdk1 (Cdk1/Clb5,6) kinase that is required for DNA replication. Deletion of Sic1 causes premature DNA replication from fewer origins, extension of the S phase and inefficient separation of sister chromatids during anaphase. Despite the well-documented relevance of Sic1 inhibition of S-Cdk1 for cell cycle control and genome instability, the molecular mechanism by which Sic1 inhibits S-Cdk1 activity remains obscure. In this paper, we show that Sic1 is functionally and structurally related to the mammalian Cki (Cdk inhibitor) p27Kip1 of the Kip/Cip family. A molecular model of the inhibitory domain of Sic1 bound to the Cdk2-cyclin A complex suggested that the yeast inhibitor might productively interface with the mammalian Cdk2-cyclin A complex. Consistent with this, Sic1 is able to bind to, and strongly inhibit the kinase activity of, the Cdk2-cyclin A complex. In addition, comparison of the different inhibitory patterns obtained using histone H1 or GST (glutathione S-transferase)-pRb (retinoblastoma protein) fusion protein as substrate (the latter of which recognizes both the docking site and the catalytic site of Cdk2-cyclin A) offers interesting suggestions for the inhibitory mechanism of Sic1. Finally, overexpression of the KIP1 gene in vivo in Saccharomyces cerevisiae, like overexpression of the related SIC1 gene, rescues the cell cycle-related phenotype of a sic1Delta strain. Taken together, these findings strongly indicate that budding yeast Sic1 and mammalian p27(Kip1) are functional homologues with a structurally conserved inhibitory domain

Density functional theory has been used to investigate structural, electronic and reactivity properties of complexes related to the peroxo forms of vanadium haloperoxidases (VHPO). In particular, the reactivity of the cofactor as a function of protonation state and environment, which are two factors thought to be crucial in modulating the activity of the enzyme, has been examined. In full agreement with experimental data, results highlight the role of protonation in the activation of the peroxo-vanadium complexes and show that the oxo-transfer step involves the unprotonated axial peroxo oxygen atom, which is easily accessible to substrates in the peroxo form of the enzyme. The role of Lys353, which in the X-ray structure of the peroxide-bound form of vanadium chloroperoxidase is hydrogen bonded to the equatorial oxygen atom of the peroxo group, has been also explored. It is concluded that Lys353 can play a role similar to a H+ in the activation of the peroxo form of the cofactor.

The cold-adapted Pseudomonas fragi lipase (PFL) displays highest activity on short-chain triglyceride substrates and is rapidly inactivated at moderate temperature. Sequence and structure comparison with homologous lipases endowed with different substrate specificity and stability, pointed to three polar residues in the lid region, that were replaced with the amino acids conserved at equivalent positions in the reference lipases. Substitutions at residues T137 and T138 modified the lipase chainlength preference profile, increasing the relative activity towards C8 substrates. Moreover, mutations conferred to PFL higher temperature stability. On the other hand, replacement of the serine at position 141 by glycine destabilized the protein.

A bridging carbonyl intermediate with key structural elements of the diiron sub-site of all-iron hydrogenase has been experimentally observed in the CN/CO substitution pathway of the {2Fe3S} carbonyl precursor, [Fe-2(CO)(5){MeSCH2C(Me)(CH2S)(2)}]. Herein we have used density functional theory (DFT) to dissect the overall substitution pathway in terms of the energetics and the structures of transition states, intermediates and products. We show that the formation of bridging CO transitions states is explicitly involved in the intimate mechanism of dicyanation. The enhanced rate of monocyanation of {2Fe3S} over the {2Fe2S} species [Fe-2(CO)(6){CH2(CH2S)(2)}] is found to rest with the ability of the thioether ligand to both stabilise a mu-CO transition state and act as a good leaving group. In contrast, the second cyanation step of the {2Fe3S} species is kinetically slower than for the {2Fe2S} monocyanide because the Fe2 atom is deactivated by coordination of the electron-donating thioether group. In addition, hindered rotation and the reaction coordinate of the approaching CN- group, are other factors which explain reactivity differences in {2Fe2S} and {2Fe3S} systems. The intermediate species formed in the second cyanation step of {2Fe3S} species is a mu-CO species, confirming the structural assignment made on the basis of FT-IR data (S. J. George, Z. Cui, M. Razavet, C. J. Pickett, Chem. Eur. J. 2002, 8, 40374046). In support of this we find that computed and experimental IR frequencies of structurally characterised {2Fe3S} species and those of the bridging carbonyl intermediate are in excellent agreement. In a wider context, the study may provide some insight into the reactivity of dinuclear systems in which neighbouring group on-off coordination plays a role in substitution pathways.

The first systematic study of diferrous dicyano dithiolates is described. Oxidation of [Fe-2(S2C2H4)(CN)(2)(CO)(4)](2-) in the presence of cyanide and tertiary phosphines and of Fe-2(S2C2H4)-(CO)(4)(PMe3)(2) in the presence of cyanide affords a series of diferrous cyanide derivatives that bear a stoichiometric, structural, and electronic relationship to the H-ox(air) state of the Fe-only hydrogenases. With PPh3 as the trapping ligand, we obtained an unsymmetrical isomer of Fe-2(S2C2H4)(mu-CO)(CN)(2)(PPh3)(2)-(CO)(2), as confirmed crystallographically. This diferrous cyanide features the semibridging CO-ligand, with Fe-mu C bond lengths of 2.15 and 1.85 angstrom. Four isomers of Fe-2(S2C2H4)(mu-CO)(CN)(2)(PMe3)(2)(CO)(2) were observed, the initial product again being unsymmetrical but more stable isomers being symmetrical. DFT calculations confirm that the most stable isomers of Fe-2(S2C2H4)(mu-CO)(CN)(2)(PMe3)(2)(CO)(2) have cyanide trans to mu-CO. Oxidative decarbonylation also afforded the new tetracyanide [Fe-2(S2C2H4)(mu-CO)(CN)(4)(CO)(2)](2-). Insights into the oxidative decarbonylation mechanism of these syntheses come from the spectroscopic characterization of the tetracarbonyl [Fe-2(S2C2H4)(mu-CO)(CN)(3)(CO)(3)](-). This species reacts with PEt3 to produce the stable adduct [Fe-2(S2C2H4)(mu-CO)(CN)(3)(CO)(2)(PEt3)](-).

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).)

Density functional theory has been used to investigate structural and electronic properties of complexes related to the resting form of the active site of vanadium haloperoxidase as a function of environment and protonation state. Results obtained by studying models of varying size and complexity highlight the influence of environment and protonation state on the structure and stability of the metal cofactor. The study shows that, in the trigonal bipyramidal active site, where one axial position is occupied by a key histidine, the trans position cannot contain a terminal oxo group. Further, a highly negatively charged vanadate unit is not stable. Protonation of at least one equatorial oxo ligand appears necessary to stabilize the metal cofactor. The study also indicates that, while at rest within the protein, the vanadate unit is most likely an anion with an axial hydroxide and an equatorial plane containing two oxos and a hydroxide. For the neutral, protonated state of the vanadate unit, there were two minima found. The first structure is characterized by an axial water with two oxo and one hydroxo group in the equatorial plane. The second structure contains an axial hydroxo group and an equatorial plane composed of one oxo and two hydroxo oxygen atoms. These two species are not significantly different in energy, indicating that either form may be important during the catalytic cycle. These data support the initial crystallographic assignment of an axially bound hydroxide, but an axial water is also a possibility. This study also shows that the protonation state of the vanadate ion is most likely greater than previously proposed.

We have applied density functional theory, using both pure (BP86) and hybrid (B3LYP and B3LYP*) functionals, to investigate structural parameters and reaction energies for nickel(II)-sulfur coordination compounds, as well as for small cluster models of the Ni-SI and Ni-R redox state of [NiFe] hydrogenases. Results obtained investigating experimentally well-characterized complexes show that BP86 is well suited to describe the structural features of this class of compounds. However, the singlet-triplet energy splitting and even the computed ground state are strongly dependent on the applied functional. Results for the cluster models of [NiFe] hydrogenases lead to the conclusion that in the reduced protein structures characterized by X-ray diffraction a hydride bridges the two metal centres. The energy splitting of the singlet and triplet states in Ni-R and Ni-SI models is calculated to be very small and may be overcome at room temperature to allow a spin crossover. Moreover, the relative stability of the Ni-SI and Ni-R structures adopted in the present investigation is fully compatible with their involvement in the reversible heterolytic cleavage of H-2.

Density functional theory has been used to investigate complexes related to the [2Fe](H) subduster of [Fe]-hydrogenases. In particular, the effects on structural and electronic properties of redox state and ligands with different sigma-donor T-acceptor character, which replace the cysteine residue coordinated to the [2Fe](H) subduster in the enzyme, have been investigated. Results show that the structural and electronic properties of fully reduced (FeFeI)-Fe-I complexes are strongly affected by the nature of the ligand L, and in particular, a progressive rotation of the Fe-d(CO)(2)(CN) group, with a CO ligand moving from a terminal to a semibridged position, is observed going from the softest to the hardest ligand. For the partially oxidized (FeFeII)-Fe-I complexes, two isomers of similar stability, characterized either by a CO ligand in a terminal or bridged position, have been observed. The switching between the two forms is associated with a spin and charge transfer between the two iron atoms, a feature that could be relevant in the catalytic mechanism of dihydrogen activation. The structure of the fully oxidized (FeFeII)-Fe-II models is extremely dependent on the nature of the L ligand; one CO group coordinated to Fed switches from terminal to bridging position going from complexes characterized by neutral to anionic L ligands

The Escherichia coli udp gene encodes uridine phosphorylase (UP), which catalyzes the reversible phosphorolysis of uridine to uracil and ribose-l-phosphate. The X-ray structure of E. coli UP resolved by two different groups produced conflicting results. In order to cast some light on the E. coli UP catalytic site, we mutagenized several residues in UP and measured by RP-HPLC the phosphorolytic activity of the mutant UP proteins in vitro. Mutations Thr94Ala, Phe162Ala, and Tyr195Gly caused a drastic decrease in UP activity. These three residues were suggested to be involved in the nucleoside binding site. However, surprisingly, Tyr195Ala caused a relative increase in enzymatic activity. Both Met197Ala and Met197Ser conserved low activity, suggesting a minor role for this residue in the UP active site. Glu196Ala completely lost UP activity, whereas the more conservative Glu196Asp, mutation was still partially active, confirming the importance of maintaining the correct charge in the surroundings of this position. Glu198 was mutated to either Gly, Asp and Gln. All three substitutions caused complete loss of enzymatic activity suggesting an important role of Glul 98 both in ribose binding and in interaction with phosphate ions. Arg30Ala and Arg91Ala eliminated UP activity, whereas Arg30Lys and Arg91Lys presented a very low activity, confirming that these residues might interact with and stabilize the phosphate ions. Ile69Ala did not decrease UP activity, whereas His8Ala lowered the activity to about 20%. Both amino acids were suggested to take part in subunit interactions. Our results confirm the structural similarity between E. coli UP and E. coli purine nucleoside phosphorylase (PNP).

The catalytic oxidation of ortho- and para-alkylthiophenols, carrying methyl or ethyl substituents at sulfur, by lactoperoxidase (LPO), horseradish peroxidase (HRP) or chloroperoxidase (CPO), in the presence of hydrogen peroxide, has been investigated. HPP and CPO are active toward these substrates, whereas LPO is only active with the ortho-substituted compounds. The enzymatic solutions containing ortho-alkylthiophenols develop an intense blue color (with optical absorptions near 400 and 600nm) that is attributed to the formation of relatively stable dimeric three-electron bonded complexes resulting from the association of enzyme-derived radical cations with the phenolic sulfide. The products of the enzymatic reactions by HRP and LPO are oligomers resulting from phenol oxidative coupling reactions and the sulfoxide, with minor amounts of oligomers containing mono or disulfoxide functionalities. With CPO the major product is always the sulfoxide, while phenol coupling products are formed in minor amounts. The selectivity exhibited by LPO toward 2- and 4-methylthiophenol has been investigated through binding experiments, NMR relaxation measurements of LPO-substrate complexes and docking calculations. The para-isomer binds much more strongly than the ortho-isomer to the enzymes. The stronger binding depends on the establishment of hydrogen bonding interactions with protein residues in the active site. (C) 2003 Elsevier Science B.V. All rights reserved.

The isomerization reaction cyanamide --> carbodiimide in vacuo and in the presence of up to six water molecules has been investigated by means of DFT and MP2 calculations, using a flexible basis set. The reliability of such methods has been checked against results of coupled cluster calculations on isolated molecules. The effect of water molecules has also been investigated in the case of the hydrolysis reaction of cyanamide and carbodiimide, leading to isourea. The number of water molecules considered is large enough to give results converged in activation and hydration energies. In addition to the water molecules explicitly described, the effect of water bulk solvent is taken into account according to the polarizable continuum model. The results show that the direct hydrolysis of H2NCN is hindered by an activation energy much higher than that for HNCNH, which in turn can be obtained from cyanamide with a relatively easy process.

Mutants F344V and F345V of Candida rugosa lipase1 (CRL1) were tested in acylation reactions of monosaccharide derivatives 1-8, in order to study the regioselectivity, and the substrate specificity of lipase variants towards unnatural substrates, such as carbohydrates. Mutant F344V showed a better reaction kinetics and/or regioselectivity then the wild type enzyme with several substrates while mutant F345V was inefficient in most cases. With the aim of correlating experimental data with the structural features of the enzyme and substrates, the interaction of substrates methyl 4,6-O-benzylidene-alpha-D-glucopyranoside (5a) and 4,6-O-benzylidene-alpha-D-galactopyranoside (6a) with the wild type enzyme and the mutant F344V was investigated, using a molecular modelling approach