RESUMO
The midpoint potential of the [Fe2S2]-Cys4-cluster in proteins is known to vary between -200 and -450 mV. This variation is caused by the different electrostatic environment of the cluster in the respective proteins. Continuum electrostatics can quantify the impact of the protein environment on the redox potential. Thus, if the redox potential of a [Fe2S2]-Cys4-cluster model compound in aqueous solution would be known, then redox potentials in various protein complexes could be calculated. However, [Fe2S2]-Cys4-cluster models are not water-soluble, and thus, their redox potential can not be measured in aqueous solution. To overcome this problem, we introduce a method that we call Virtual Model Compound Approach (VMCA) to extrapolate the model redox potential from known redox potentials of proteins. We carefully selected high-resolution structures for our analysis and divide them into a fit set, for fitting the model redox potential, and an independent test set, to check the validity of the model redox potential. However, from our analysis, we realized that the some structures can not be used as downloaded from the PDB but had to be re-refined in order to calculate reliable redox potentials. Because of the re-refinement, we were able to significantly reduce the standard deviation of our derived model redox potential for the [Fe2S2]-Cys4-cluster from 31 mV to 10 mV. As the model redox potential, we obtained -184 mV. This model redox potential can be used to analyze the redox behavior of [Fe2S2]-Cys4-clusters in larger protein complexes.
Assuntos
Proteínas Ferro-Enxofre , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , OxirreduçãoRESUMO
The handling of plastic waste and the associated ubiquitous occurrence of microplastic poses one of the biggest challenges of our time. Recent investigations of plastic degrading enzymes have opened new prospects for biological microplastic decomposition as well as recycling applications. For polyethylene terephthalate, in particular, several natural and engineered enzymes are known to have such promising properties. From a previous study that identified new PETase candidates by homology search, we chose the candidate PET6 from the globally distributed, halophilic organism Vibrio gazogenes for further investigation. By mapping the occurrence of Vibrios containing PET6 homologs we demonstrated their ubiquitous prevalence in the pangenome of several Vibrio strains. The biochemical characterization of PET6 showed that PET6 has a comparatively lower activity than other enzymes but also revealed a superior turnover at very high salt concentrations. The crystal structure of PET6 provides structural insights into this adaptation to saline environments. By grafting only a few beneficial mutations from other PET degrading enzymes onto PET6, we increased the activity up to three-fold, demonstrating the evolutionary potential of the enzyme. MD simulations of the variant helped rationalize the mutational effects of those mutants and elucidate the interaction of the enzyme with a PET substrate. With tremendous amounts of plastic waste in the Ocean and the prevalence of Vibrio gazogenes in marine biofilms and estuarine marshes, our findings suggest that Vibrio and the PET6 enzyme are worthy subjects to study the PET degradation in marine environments.
Assuntos
Hidrolases , Vibrio , Humanos , Hidrolases/química , Plásticos , Microplásticos , Vibrio/genéticaRESUMO
The catalytic mechanisms of serine and cysteine peptidases are similar: the proton of the nucleophile (serine or cysteine) is transferred to the catalytic histidine, and the nucleophile attacks the substrate for cleavage. However, they differ in an important aspect: cysteine peptidases form a stable ion-pair intermediate in a stepwise mechanism, while serine peptidases follow a concerted mechanism. While it is known that a positive electrostatic potential at the active site of cysteine peptidases stabilizes the cysteine anion in the ion-pair state, the physical basis of the concerted mechanism of serine peptidases is poorly understood. In this work, we use continuum electrostatic analysis and quantum mechanical/molecular mechanical (QM/MM) simulations to demonstrate that a destabilization of an anionic serine by a negative electrostatic potential in combination with a compact active site geometry facilitates a concerted mechanism in serine peptidases. Moreover, we show that an anionic serine would destabilize the protein significantly compared to an anionic cysteine in cysteine peptidases, which underlines the necessity of a concerted mechanism for serine peptidases. On the basis of our calculations on an inactive serine mutant of a natural cysteine peptidase, we show that the energy barrier for the catalytic mechanism can be substantially decreased by introducing a negative electrostatic potential and by reducing the relevant distances indicating that these parameters are essential for the activity of serine peptidases. Our work demonstrates that the concerted mechanism of serine peptidases represents an evolutionary innovative way to perform catalysis without the energetically expensive need to stabilize the anionic serine. In contrast in cysteine peptidases, the anionic cysteine is energetically easily accessible and it is a very efficient nucleophile, making these peptidases mechanistically simple. However, a cysteine is highly oxygen sensitive, which is problematic in an aerobic environment. On the basis of the analysis in this work, we suggest that serine peptidases represent an oxygen-insensitive alternative to cysteine peptidases.
Assuntos
Cisteína , Serina , Catálise , Domínio Catalítico , Cisteína/química , Oxigênio , Serina Endopeptidases/metabolismo , Eletricidade EstáticaRESUMO
Phytochelatins (PCs) are nonribosomal thiol-rich oligopeptides synthetized from glutathione (GSH) in a γ-glutamylcysteinyl transpeptidation reaction catalyzed by PC synthases (PCSs). Ubiquitous in plant and present in some invertebrates, PCSs are involved in metal detoxification and homeostasis. The PCS-like enzyme from the cyanobacterium Nostoc sp. (NsPCS) is considered to be an evolutionary precursor enzyme of genuine PCSs because it shows sufficient sequence similarity for homology to the catalytic domain of the eukaryotic PCSs and shares the peptidase activity consisting in the deglycination of GSH. In this work, we investigate the catalytic mechanism of NsPCS by combining structural, spectroscopic, thermodynamic, and theoretical techniques. We report several crystal structures of NsPCS capturing different states of the catalyzed chemical reaction: (i) the structure of the wild-type enzyme (wt-NsPCS); (ii) the high-resolution structure of the γ-glutamyl-cysteine acyl-enzyme intermediate (acyl-NsPCS); and (iii) the structure of an inactive variant of NsPCS, with the catalytic cysteine mutated into serine (C70S-NsPCS). We characterize NsPCS as a relatively slow enzyme whose activity is sensitive to the redox state of the substrate. Namely, NsPCS is active with reduced glutathione (GSH), but is inhibited by oxidized glutathione (GSSG) because the cleavage product is not released from the enzyme. Our biophysical analysis led us to suggest that the biological function of NsPCS is being a part of a redox sensing system. In addition, we propose a mechanism how PCS-like enzymes may have evolved toward genuine PCS enzymes.
Assuntos
Aminoaciltransferases , Nostoc , Aminoaciltransferases/metabolismo , Cisteína/metabolismo , Glutationa/química , Nostoc/metabolismo , Oxirredução , Peptídeo Hidrolases , Fitoquelatinas/metabolismoRESUMO
In lichen symbioses, fungal secondary metabolites provide UV protection on which lichen algae such as trebouxiophycean green algae-the most prominent group of photobionts in lichen symbioses-sensitively depend. These metabolites differ in their UV absorbance capability and solvability, and thus vary in their propensity of being leached from the lichen body in humid and warm environments, with still unknown implications for the global distribution of lichens. In this study covering more than 10,000 lichenised fungal species, we show that the occurrence of fungal-derived metabolites in combination with their UV absorbance capability and their probability of being leached in warm and humid environments are important eco-evolutionary drivers of global lichen distribution. Fungal-derived UV protection seems to represent an indirect environmental adaptation in which the lichen fungus invests to protect the trebouxiophycean photobiont from high UV radiation in warm and humid climates and, by doing this, secures its carbon source.
Assuntos
Clorófitas , Líquens , Evolução Biológica , Clima , Filogenia , SimbioseRESUMO
Optimal charge distribution is considered to be important for efficient formation of protein complexes. Electrostatic interactions guide encounter complex formation that precedes the formation of an active protein complex. However, disturbing the optimized distribution by introduction of extra charged patches on cytochrome c peroxidase does not lead to a reduction in productive encounters with its partner cytochrome c. To test whether a complex with a high population of encounter complex is more easily affected by suboptimal charge distribution, the interactions of cytochrome c mutant R13A with wild-type cytochrome c peroxidase and a variant with an additional negative patch were studied. The complex of the peroxidase and cytochrome c R13A was reported to have an encounter state population of 80%, compared to 30% for the wild-type cytochrome c. NMR analysis confirms the dynamic nature of the interaction and demonstrates that the mutant cytochrome c samples the introduced negative patch. Kinetic experiments show that productive complex formation is fivefold to sevenfold slower at moderate and high ionic strength values for cytochrome c R13A but the association rate is not affected by the additional negative patch on cytochrome c peroxidase, showing that the total charge on the protein surface can compensate for less optimal charge distribution. At low ionic strength (44 mm), the association with the mutant cytochrome c reaches the same high rates as found for wild-type cytochrome c, approaching the diffusion limit.
Assuntos
Citocromo-c Peroxidase/genética , Complexos Multiproteicos/genética , Conformação Proteica , Citocromo-c Peroxidase/ultraestrutura , Transporte de Elétrons/genética , Cinética , Modelos Moleculares , Método de Monte Carlo , Complexos Multiproteicos/ultraestrutura , Concentração Osmolar , Saccharomyces cerevisiae/genética , Eletricidade EstáticaRESUMO
The water-soluble quencher hydrodabcyl can be activated as an N-succinimidyl ester that is readily accessible from crude hydrodabcyl and storable for a long time. With primary and secondary amines, it reacts swiftly and chemoselectively, even in the presence of other competing nucleophiles such as those typically present in natural peptides. One of the three phenolic OH groups of hydrodabcyl is amenable to selective mono-Boc protection resulting in reduced polarity, advantageous to its further use in organic synthesis. The advantages of hydrodabcyl over dabcyl in spectrometric applications are exemplified by the pH dependence of its absorbance spectra.
RESUMO
Protein complex formation depends strongly on electrostatic interactions. The distribution of charges on the surface of redox proteins is often optimized by evolution to guide recognition and binding. To test the degree to which the electrostatic interactions between cytochrome c peroxidase (CcP) and cytochrome c (Cc) are optimized, we produced five CcP variants, each with a different charge distribution on the surface. Monte Carlo simulations show that the addition of negative charges attracts Cc to the new patches, and the neutralization of the charges in the regular, stereospecific binding site for Cc abolishes the electrostatic interactions in that region entirely. For CcP variants with the charges in the regular binding site intact, additional negative patches slightly enhance productive complex formation, despite disrupting the optimized charge distribution. Removal of the charges in the regular binding site results in a dramatic decrease in the complex formation rate, even in the presence of highly negative patches elsewhere on the surface. We conclude that additional charge patches can result in either productive or futile encounter complexes, depending on whether negative residues are located also in the regular binding site.
Assuntos
Citocromo-c Peroxidase/química , Citocromos c/química , Método de Monte Carlo , Saccharomyces cerevisiae/metabolismo , Eletricidade Estática , Sítios de Ligação , Citocromo-c Peroxidase/metabolismo , Citocromos c/metabolismo , Transporte de Elétrons , Modelos Moleculares , Oxirredução , Conformação ProteicaRESUMO
Electrostatic interactions can strongly increase the efficiency of protein complex formation. The charge distribution in redox proteins is often optimized to steer a redox partner to the electron transfer active binding site. To test whether the optimized distribution is more important than the strength of the electrostatic interactions, an additional negative patch was introduced on the surface of cytochromeâ c peroxidase, away from the stereospecific binding site, and its effect on the encounter complex as well as the rate of complex formation was determined. Monte Carlo simulations and paramagnetic relaxation enhancement NMR experiments indicate that the partner, cytochromeâ c, interacts with the new patch. Unexpectedly, the rate of the active complex formation was not reduced, but rather slightly increased. The findings support the idea that for efficient protein complex formation the strength of the electrostatic interaction is more critical than an optimized charge distribution.
Assuntos
Citocromo-c Peroxidase/metabolismo , Sítios de Ligação , Citocromo-c Peroxidase/química , Transporte de Elétrons , Simulação de Dinâmica Molecular , Método de Monte Carlo , Conformação Proteica , Saccharomyces cerevisiae/enzimologia , Eletricidade EstáticaRESUMO
We use real-time density functional theory on a real-space grid to calculate electronic excitations of bacteriochlorophyll chromophores of the light-harvesting complex 2 (LH2). Comparison with Gaussian basis set calculations allows us to assess the numerical trust range for computing electron dynamics in coupled chromophores with both types of techniques. Tuned range-separated hybrid calculations for one bacteriochlorophyll as well as two coupled ones are used as a reference against which we compare results from the adiabatic time-dependent local density approximation (TDLDA). The tuned range-separated hybrid calculations lead to a qualitatively correct description of the electronic excitations and couplings. They allow us to identify spurious charge-transfer excitations that are obtained with the TDLDA. When we take into account the environment that the LH2 protein complex forms for the bacteriochlorophylls, we find that it substantially shifts the energy of the spurious charge-transfer excitations, restoring a qualitatively correct electronic coupling of the dominant excitations also for TDLDA.
Assuntos
Bacterioclorofilas/química , Complexos de Proteínas Captadores de Luz/química , Beijerinckiaceae/química , Teoria da Densidade Funcional , Transferência de Energia , Modelos QuímicosRESUMO
Hydride transfers play a crucial role in a multitude of biological redox reactions and are mediated by flavin, deazaflavin or nicotinamide adenine dinucleotide cofactors at standard redox potentials ranging from 0 to -340 mV. 2-Naphthoyl-CoA reductase, a key enzyme of oxygen-independent bacterial naphthalene degradation, uses a low-potential one-electron donor for the two-electron dearomatization of its substrate below the redox limit of known biological hydride transfer processes at E°' = -493 mV. Here we demonstrate by X-ray structural analyses, QM/MM computational studies, and multiple spectroscopy/activity based titrations that highly cooperative electron transfer (n = 3) from a low-potential one-electron (FAD) to a two-electron (FMN) transferring flavin cofactor is the key to overcome the resonance stabilized aromatic system by hydride transfer in a highly hydrophobic pocket. The results evidence how the protein environment inversely functionalizes two flavins to switch from low-potential one-electron to hydride transfer at the thermodynamic limit of flavin redox chemistry.
Assuntos
Proteínas de Bactérias/química , Coenzimas/química , Flavinas/química , Modelos Moleculares , Oxirredutases/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Coenzimas/metabolismo , Simulação por Computador , Cristalografia por Raios X , Transporte de Elétrons , Flavinas/metabolismo , Naftalenos/química , Naftalenos/metabolismo , Oxirredutases/isolamento & purificação , Oxirredutases/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise EspectralRESUMO
MCMap is a tool particularly well-suited for analyzing energy landscapes of transient macromolecular complexes. The program applies a Monte Carlo strategy, where the ligand moves randomly in the electrostatic field of the receptor. By applying importance sampling, the major interaction sites are mapped, resulting in a global distribution of ligand-receptor complexes. This approach displays the dynamic character of transiently interacting protein complexes where not a single complex but an ensemble of complexes better describes the protein interactions. The software provides a broad range of analysis options which allow for relating the simulations to experimental data and for interpreting them on a structural level. The application of MCMap is exemplified by the electron-transfer complex of cytochrome c peroxidase and cytochrome c from baker's yeast. The functionality of MCMap and the visualization of simulation data are in particular demonstrated by studying the dependence of the association on ionic strength and on the oxidation state of the binding partner. Furthermore, microscopically, a repulsion of a second ligand can be seen in the ternary complex upon the change of the oxidation state of the bound cytochrome c. The software is made available as open source software together with the example and can be downloaded free of charge from http://www.bisb.uni-bayreuth.de/index.php?page=downloads.
RESUMO
Aromatic compounds are environmental pollutants with toxic and carcinogenic properties. Despite the stability of aromatic rings, bacteria are able to degrade the aromatic compounds into simple metabolites and use them as growth substrates under oxic or even under anoxic conditions. In anaerobic microorganisms, most monocyclic aromatic growth substrates are converted to the central intermediate benzoyl-coenzyme A, which is enzymatically reduced to cyclohexa-1,5-dienoyl-CoA. The strictly anaerobic bacterium Geobacter metallireducens uses the class II benzoyl-CoA reductase complex for this reaction. The catalytic BamB subunit of this complex harbors an active site tungsten-bis-pyranopterin cofactor with the metal being coordinated by five protein/cofactor-derived sulfur atoms and a sixth, so far unknown, ligand. Although BamB has been biochemically and structurally characterized, its mechanism still remains elusive. Here we use continuum electrostatic and QM/MM calculations to model benzoyl-CoA reduction by BamB. We aim to elucidate the identity of the sixth ligand of the active-site tungsten ion together with the interplay of the electron and proton transfer events during the aromatic ring reduction. On the basis of our calculations, we propose that benzoyl-CoA reduction is initiated by a hydrogen atom transfer from a W(IV) species with an aqua ligand, yielding W(V)-[OH-] and a substrate radical intermediate. In the next step, a proton-assisted second electron transfer takes place with a conserved active-site histidine serving as the second proton donor. Interestingly, our calculations suggest that the electron for the second reduction step is taken from the pyranopterin cofactors rather than from the tungsten ion. The resulting cationic radical, which is distributed over both pyranopterins, is stabilized by conserved anionic amino acid residues. The stepwise mechanism of the reduction shows similarities to the Birch reduction known from organic chemistry. However, the strict coupling of protons and electrons allows the reaction to proceed under milder conditions.
Assuntos
Benzeno/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Tungstênio/metabolismo , Acil Coenzima A/metabolismo , Domínio Catalítico , Transporte de Elétrons , Geobacter/enzimologia , Histidina/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Prótons , Pterinas/metabolismo , Teoria QuânticaRESUMO
Phenazines are bacterial virulence and survival factors with important roles in infectious disease. PhzF catalyzes a key reaction in their biosynthesis by isomerizing (2 S,3 S)-2,3-dihydro-3-hydroxy anthranilate (DHHA) in two steps, a [1,5]-hydrogen shift followed by tautomerization to an aminoketone. While the [1,5]-hydrogen shift requires the conserved glutamate E45, suggesting acid/base catalysis, it also shows hallmarks of a sigmatropic rearrangement, namely the suprafacial migration of a non-acidic proton. To discriminate these mechanistic alternatives, we employed enzyme kinetic measurements and computational methods. Quantum mechanics/molecular mechanics (QM/MM) calculations revealed that the activation barrier of a proton shuttle mechanism involving E45 is significantly lower than that of a sigmatropic [1,5]-hydrogen shift. QM/MM also predicted a large kinetic isotope effect, which was indeed observed with deuterated substrate. For the tautomerization, QM/MM calculations suggested involvement of E45 and an active site water molecule, explaining the observed stereochemistry. Because these findings imply that PhzF can act only on a limited substrate spectrum, we also investigated the turnover of DHHA derivatives, of which only O-methyl and O-ethyl DHHA were converted. Together, these data reveal how PhzF orchestrates a water-free with a water-dependent step. Its unique mechanism, specificity and essential role in phenazine biosynthesis may offer opportunities for inhibitor development.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Fenazinas/metabolismo , Pseudomonas fluorescens/metabolismo , Catálise , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Pseudomonas fluorescens/crescimento & desenvolvimento , Teoria Quântica , Especificidade por SubstratoRESUMO
Understanding enzyme mechanisms is a major task to achieve in order to comprehend how living cells work. Recent advances in biomolecular research provide huge amount of data on enzyme kinetics and structure. The analysis of diverse experimental results and their combination into an overall picture is, however, often challenging. Microscopic details of the enzymatic processes are often anticipated based on several hints from macroscopic experimental data. Computational biochemistry aims at creation of a computational model of an enzyme in order to explain microscopic details of the catalytic process and reproduce or predict macroscopic experimental findings. Results of such computations are in part complementary to experimental data and provide an explanation of a biochemical process at the microscopic level. In order to evaluate the mechanism of an enzyme, a structural model is constructed which can be analyzed by several theoretical approaches. Several simulation methods can and should be combined to get a reliable picture of the process of interest. Furthermore, abstract models of biological systems can be constructed combining computational and experimental data. In this review, we discuss structural computational models of enzymatic systems. We first discuss various models to simulate enzyme catalysis. Furthermore, we review various approaches how to characterize the enzyme mechanism both qualitatively and quantitatively using different modeling approaches.
Assuntos
Biologia Computacional/métodos , Enzimas/metabolismo , Termodinâmica , Animais , Bioquímica/métodos , Enzimas/química , Humanos , Cinética , Simulação de Dinâmica Molecular , Teoria QuânticaRESUMO
In this work, we calculate the protonation probabilities of titratable residues of bovine rhodopsin using the Poisson-Boltzmann equation. We also consider the influence of the membrane potential. Our results indicate that at physiological pH, the titratable groups directly involved in photosensing, namely Glu113, Glu181 and the retinal Schiff base, are charged. In contrast, the residues Asp83, Glu122 and His211, which are buried in the membrane, are uncharged. However, as these later residues are localized in the middle of the membrane, they are exposed to the membrane potential more strongly, which may have important functional implications. Despite of their large distance, Asp83 and Glu122 interact relatively strongly. As these two residues are in contact with opposite sides of the membrane, the membrane potential has different effects on them, which allows an enhancement of the membrane potential signal. An analysis of the different contributions to the protonation energy indicates that conformational changes that reduce the desolvation penalty of Asp83, Glu122 and His211 may lead to a complex protonation pattern change that allows an influence of the membrane potential on the function of rhodopsin. The high degree of evolutionary conservation of these three buried residues supports the idea of their functional importance. Our results are in-line with many experimental findings and lead to new ideas that can be experimentally tested.
Assuntos
Prótons , Rodopsina/química , Animais , Bovinos , Simulação por Computador , Concentração de Íons de Hidrogênio , Potenciais da Membrana , Eletricidade EstáticaRESUMO
His-tag technology is employed to bind membrane proteins, such as the bc1 complex and the reaction center (RC) from Rhodobacter sphaeroides, to spherical as well as planar surfaces in a strict orientation. Subsequently, the spherical and planar surfaces are subjected to in situ dialysis to form proteo-lipobeads (PLBs) and protein-tethered bilayer membranes, respectively. PLBs based on Ni-nitrileotriacetic acid-functionalized agarose beads that have diameters ranging from 50 to 150 µm are used to assess proton release and membrane potential parameters by confocal laser-scanning microscopy. The pH and potential transients are thus obtained from bc1 activated by the RC. To assess the turnover of bc1 excited by the RC in a similar setting, we used the planar surface of an attenuated total reflection crystal modified with a thin gold layer to carry out time-resolved surface-enhanced IR absorption spectroscopy triggered by flash lamp excitation. The experiments suggest that both proteins interact in a cyclic manner in both environments. The activity of the proteins seems to be preserved in the same manner as that in chromatophores or reconstituted in liposomes.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Rhodobacter sphaeroides/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Tamanho da Partícula , Rhodobacter sphaeroides/química , Propriedades de SuperfícieRESUMO
Conjugate peak refinement (CPR) is a powerful and robust method to search transition states on a molecular potential energy surface. Nevertheless, the method was to the best of our knowledge so far only implemented in CHARMM. In this paper, we present PyCPR, a new Python-based implementation of the CPR algorithm within the pDynamo framework. We provide a detailed description of the theory underlying our implementation and discuss the different parts of the implementation. The method is applied to two different problems. First, we illustrate the method by analyzing the gauche to anti-periplanar transition of butane using a semiempirical QM method. Second, we reanalyze the mechanism of a glycyl-radical enzyme, namely of 4-hydroxyphenylacetate decarboxylase (HPD) using QM/MM calculations. In the end, we suggest a strategy how to use our implementation of the CPR algorithm. The integration of PyCPR into the framework pDynamo allows the combination of CPR with the large variety of methods implemented in pDynamo. PyCPR can be used in combination with quantum mechanical and molecular mechanical methods (and hybrid methods) implemented directly in pDynamo, but also in combination with external programs such as ORCA using pDynamo as interface. PyCPR is distributed as free, open source software and can be downloaded from http://www.bisb.uni-bayreuth.de/index.php?page=downloads . Graphical Abstract PyCPR is a search tool for finding saddle points on the potential energy landscape of a molecular system.
RESUMO
In biology, tungsten (W) is exclusively found in microbial enzymes bound to a bis-pyranopterin cofactor (bis-WPT). Previously known W enzymes catalyze redox oxo/hydroxyl transfer reactions by directly coordinating their substrates or products to the metal. They comprise the W-containing formate/formylmethanofuran dehydrogenases belonging to the dimethyl sulfoxide reductase (DMSOR) family and the aldehyde:ferredoxin oxidoreductase (AOR) families, which form a separate enzyme family within the Mo/W enzymes. In the last decade, initial insights into the structure and function of two unprecedented W enzymes were obtained: the acetaldehyde forming acetylene hydratase (ACH) belongs to the DMSOR and the class II benzoyl-coenzyme A (CoA) reductase (BCR) to the AOR family. The latter catalyzes the reductive dearomatization of benzoyl-CoA to a cyclic diene. Both are key enzymes in the degradation of acetylene (ACH) or aromatic compounds (BCR) in strictly anaerobic bacteria. They are unusual in either catalyzing a nonredox reaction (ACH) or a redox reaction without coordinating the substrate or product to the metal (BCR). In organic chemical synthesis, analogous reactions require totally nonphysiological conditions depending on Hg2+ (acetylene hydration) or alkali metals (benzene ring reduction). The structural insights obtained pave the way for biological or biomimetic approaches to basic reactions in organic chemistry.
Assuntos
Hidroliases/química , Hidroliases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Tungstênio/metabolismo , Bactérias Anaeróbias/enzimologia , Bactérias Anaeróbias/metabolismo , Domínio Catalítico , Coenzimas/química , Coenzimas/metabolismo , Computadores Moleculares , Hidrocarbonetos Aromáticos/metabolismo , Modelos Moleculares , Oxirredução , Filogenia , Estrutura Terciária de Proteína , Pterinas/metabolismoRESUMO
4-Hydroxyphenylacetate decarboxylase (4Hpad) is the prototype of a new class of Fe-S cluster-dependent glycyl radical enzymes (Fe-S GREs) acting on aromatic compounds. The two-enzyme component system comprises a decarboxylase responsible for substrate conversion and a dedicated activating enzyme (4Hpad-AE). The decarboxylase uses a glycyl/thiyl radical dyad to convert 4-hydroxyphenylacetate into p-cresol (4-methylphenol) by a biologically unprecedented Kolbe-type decarboxylation. In addition to the radical dyad prosthetic group, the decarboxylase unit contains two [4Fe-4S] clusters coordinated by an extra small subunit of unknown function. 4Hpad-AE reductively cleaves S-adenosylmethionine (SAM or AdoMet) at a site-differentiated [4Fe-4S]2+/+ cluster (RS cluster) generating a transient 5'-deoxyadenosyl radical that produces a stable glycyl radical in the decarboxylase by the abstraction of a hydrogen atom. 4Hpad-AE binds up to two auxiliary [4Fe-4S] clusters coordinated by a ferredoxin-like insert that is C-terminal to the RS cluster-binding motif. The ferredoxin-like domain with its two auxiliary clusters is not vital for SAM-dependent glycyl radical formation in the decarboxylase, but facilitates a longer lifetime for the radical. This review describes the 4Hpad and cognate AE families and focuses on the recent advances and open questions concerning the structure, function and mechanism of this novel Fe-S-dependent class of GREs.