RESUMO
To acquire essential Fe(III), bacteria produce and secrete siderophores with high affinity and selectivity for Fe(III) to mediate its uptake into the cell. Here, we show that the periplasmic binding protein CeuE of Campylobacter jejuni, which was previously thought to bind the Fe(III) complex of the hexadentate siderophore enterobactin (Kd â¼ 0.4 ± 0.1 µM), preferentially binds the Fe(III) complex of the tetradentate enterobactin hydrolysis product bis(2,3-dihydroxybenzoyl-l-Ser) (H5-bisDHBS) (Kd = 10.1 ± 3.8 nM). The protein selects Λ-configured [Fe(bisDHBS)](2-) from a pool of diastereomeric Fe(III)-bisDHBS species that includes complexes with metal-to-ligand ratios of 1:1 and 2:3. Cocrystal structures show that, in addition to electrostatic interactions and hydrogen bonding, [Fe(bisDHBS)](2-) binds through coordination of His227 and Tyr288 to the iron center. Similar binding is observed for the Fe(III) complex of the bidentate hydrolysis product 2,3-dihydroxybenzoyl-l-Ser, [Fe(monoDHBS)2](3-) The mutation of His227 and Tyr288 to noncoordinating residues (H227L/Y288F) resulted in a substantial loss of affinity for [Fe(bisDHBS)](2-) (Kd â¼ 0.5 ± 0.2 µM). These results suggest a previously unidentified role for CeuE within the Fe(III) uptake system of C. jejuni, provide a molecular-level understanding of the underlying binding pocket adaptations, and rationalize reports on the use of enterobactin hydrolysis products by C. jejuni, Vibrio cholerae, and other bacteria with homologous periplasmic binding proteins.
Assuntos
Proteínas de Bactérias/química , Campylobacter jejuni/metabolismo , Proteínas de Transporte/química , Complexos de Coordenação/química , Enterobactina/metabolismo , Ferro/metabolismo , Sideróforos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzoatos/química , Benzoatos/metabolismo , Campylobacter jejuni/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Complexos de Coordenação/metabolismo , Cristalografia por Raios X , Expressão Gênica , Hidrazonas/química , Hidrazonas/metabolismo , Ligação de Hidrogênio , Hidrólise , Transporte de Íons , Proteínas de Ligação ao Ferro , Ligantes , Modelos Moleculares , Mutação , Ligação Proteica , Eletricidade Estática , EstereoisomerismoRESUMO
Amylases are probably the best studied glycoside hydrolases and have a huge biotechnological value for industrial processes on starch. Multiple amylases from fungi and microbes are currently in use. Whereas bacterial amylases are well suited for many industrial processes due to their high stability, fungal amylases are recognized as safe and are preferred in the food industry, although they lack the pH tolerance and stability of their bacterial counterparts. Here, we describe three amylases, two of which have a broad pH spectrum extending to pH 8 and higher stability well suited for a broad set of industrial applications. These enzymes have the characteristic GH13 α-amylase fold with a central (ß/α)8-domain, an insertion domain with the canonical calcium binding site and a C-terminal ß-sandwich domain. The active site was identified based on the binding of the inhibitor acarbose in form of a transglycosylation product, in the amylases from Thamnidium elegans and Cordyceps farinosa. The three amylases have shortened loops flanking the nonreducing end of the substrate binding cleft, creating a more open crevice. Moreover, a potential novel binding site in the C-terminal domain of the Cordyceps enzyme was identified, which might be part of a starch interaction site. In addition, Cordyceps farinosa amylase presented a successful example of using the microseed matrix screening technique to significantly speed-up crystallization.
Assuntos
Amilases/química , Amilases/metabolismo , Fungos/enzimologia , Sítios de Ligação , Domínio Catalítico , Ativação Enzimática , Estabilidade Enzimática , Glucose/química , Glucose/metabolismo , Glicosilação , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Relação Estrutura-Atividade , alfa-Amilases/química , alfa-Amilases/metabolismoRESUMO
Here, we present a lipase mutant containing a biochemical switch allowing a controlled opening and closing of the lid independent of the environment. The closed form of the TlL mutant shows low binding to hydrophobic surfaces compared to the binding observed after activating the controlled switch inducing lid-opening. We directly show that lipid binding of this mutant is connected to an open lid conformation demonstrating the impact of the exposed amino acid residues and their participation in binding at the water-lipid interface. The switch was created by introducing two cysteine residues into the protein backbone at sites 86 and 255. The crystal structure of the mutant shows the successful formation of a disulfide bond between C86 and C255 which causes strained closure of the lid-domain. Control of enzymatic activity and binding was demonstrated on substrate emulsions and natural lipid layers. The locked form displayed low enzymatic activity (~10%) compared to wild-type. Upon release of the lock, enzymatic activity was fully restored. Only 10% binding to natural lipid substrates was observed for the locked lipase compared to wild-type, but binding was restored upon adding reducing agent. QCM-D measurements revealed a seven-fold increase in binding rate for the unlocked lipase. The TlL_locked mutant shows structural changes across the protein important for understanding the mechanism of lid-opening and closing. Our experimental results reveal sites of interest for future mutagenesis studies aimed at altering the activation mechanism of TlL and create perspectives for generating tunable lipases that activate under controlled conditions.
Assuntos
Ascomicetos/enzimologia , Lipase/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lipase/química , Conformação Proteica , Engenharia de Proteínas , Espectrometria de Fluorescência , Especificidade por SubstratoRESUMO
A microcrystalline suspension of Bacillus lentus subtilisin (Savinase) produced during industrial large-scale production was analysed by X-ray powder diffraction (XRPD) and X-ray single-crystal diffraction (MX). XRPD established that the bulk microcrystal sample representative of the entire production suspension corresponded to space group P212121, with unit-cell parameters a = 47.65, b = 62.43, c = 75.74â Å, equivalent to those for a known orthorhombic crystal form (PDB entry 1ndq). MX using synchrotron beamlines at the Diamond Light Source with beam dimensions of 20 × 20â µm was subsequently used to study the largest crystals present in the suspension, with diffraction data being collected from two single crystals (â¼20 × 20 × 60â µm) to resolutions of 1.40 and 1.57â Å, respectively. Both structures also belonged to space group P2(1)2(1)2(1), but were quite distinct from the dominant form identified by XRPD, with unit-cell parameters a = 53.04, b = 57.55, c = 71.37â Å and a = 52.72, b = 57.13, c = 65.86â Å, respectively, and refined to R = 10.8% and Rfree = 15.5% and to R = 14.1% and Rfree = 18.0%, respectively. They are also different from any of the forms previously reported in the PDB. A controlled crystallization experiment with a highly purified Savinase sample allowed the growth of single crystals of the form identified by XRPD; their structure was solved and refined to a resolution of 1.17â Å with an R of 9.2% and an Rfree of 11.8%. Thus, there are at least three polymorphs present in the production suspension, albeit with the 1ndq-like microcrystals predominating. It is shown how the two techniques can provide invaluable and complementary information for such a production suspension and it is proposed that XRPD provides an excellent quality-control tool for such suspensions.
Assuntos
Bacillus/enzimologia , Difração de Pó/métodos , Subtilisina/química , Microscopia de Força Atômica , Modelos Moleculares , Estrutura Terciária de Proteína , Subtilisina/análiseRESUMO
Iron-bound structure: The ferric complex of a tetradentate siderophore mimic was synthesized and co-crystallized with the periplasmic binding protein CeuE of Campylobacterâ jejuni. In addition to electrostatic and hydrogen-bonding interactions between the binding pocket and the substrate, the structure showed direct coordination of two amino acid side chains to the Fe(III) center (orange, see figure).
Assuntos
Complexos de Coordenação/química , Proteínas Periplásmicas de Ligação/química , Sideróforos/química , Sequência de Aminoácidos , Materiais Biomiméticos/química , Modelos MolecularesRESUMO
Many secreted eukaryotic proteins are N-glycosylated with oligosaccharides composed of a high-mannose N-glycan core and, in the specific case of yeast cell-wall proteins, an extended α-1,6-mannan backbone carrying a number of α-1,2- and α-1,3-mannose substituents of varying lengths. α-Mannosidases from CAZy family GH92 release terminal mannose residues from these N-glycans, providing access for the α-endomannanases, which then degrade the α-mannan backbone. Most characterized GH92 α-mannosidases consist of a single catalytic domain, while a few have extra domains including putative carbohydrate-binding modules (CBMs). To date, neither the function nor the structure of a multi-domain GH92 α-mannosidase CBM has been characterized. Here, the biochemical investigation and crystal structure of the full-length five-domain GH92 α-1,2-mannosidase from Neobacillus novalis (NnGH92) with mannoimidazole bound in the active site and an additional mannoimidazole bound to the N-terminal CBM32 are reported. The structure of the catalytic domain is very similar to that reported for the GH92 α-mannosidase Bt3990 from Bacteroides thetaiotaomicron, with the substrate-binding site being highly conserved. The function of the CBM32s and other NnGH92 domains was investigated by their sequential deletion and suggested that whilst their binding to the catalytic domain was crucial for the overall structural integrity of the enzyme, they appear to have little impact on the binding affinity to the yeast α-mannan substrate. These new findings provide a better understanding of how to select and optimize other multi-domain bacterial GH92 α-mannosidases for the degradation of yeast α-mannan or mannose-rich glycans.
Assuntos
Mananas , Manosidases , Manosidases/química , Manosidases/metabolismo , alfa-Manosidase/metabolismo , Mananas/química , Mananas/metabolismo , Manose/química , Manose/metabolismo , Saccharomyces cerevisiae/metabolismo , Modelos Moleculares , Polissacarídeos/química , Especificidade por SubstratoRESUMO
Muramidases (also known as lysozymes) hydrolyse the peptidoglycan component of the bacterial cell wall and are found in many glycoside hydrolase (GH) families. Similar to other glycoside hydrolases, muramidases sometimes have noncatalytic domains that facilitate their interaction with the substrate. Here, the identification, characterization and X-ray structure of a novel fungal GH24 muramidase from Trichophaea saccata is first described, in which an SH3-like cell-wall-binding domain (CWBD) was identified by structure comparison in addition to its catalytic domain. Further, a complex between a triglycine peptide and the CWBD from T. saccata is presented that shows a possible anchor point of the peptidoglycan on the CWBD. A `domain-walking' approach, searching for other sequences with a domain of unknown function appended to the CWBD, was then used to identify a group of fungal muramidases that also contain homologous SH3-like cell-wall-binding modules, the catalytic domains of which define a new GH family. The properties of some representative members of this family are described as well as X-ray structures of the independent catalytic and SH3-like domains of the Kionochaeta sp., Thermothielavioides terrestris and Penicillium virgatum enzymes. This work confirms the power of the module-walking approach, extends the library of known GH families and adds a new noncatalytic module to the muramidase arsenal.
Assuntos
Muramidase , Peptidoglicano , Muramidase/química , Sequência de Aminoácidos , Modelos Moleculares , Glicosídeo Hidrolases/química , Parede CelularRESUMO
Members of the Leishmania genus are the causative agents of the life-threatening disease leishmaniasis. New drugs are being sought due to increasing resistance and adverse side effects with current treatments. The knowledge that dUTPase is an essential enzyme and that the all α-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric ß-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding. These findings will aid in the development of dUTPase inhibitors as potential new lead anti-trypanosomal compounds.
Assuntos
Nucleotídeos de Desoxiuracil/química , Desoxiuridina/química , Leishmania major/enzimologia , Multimerização Proteica , Proteínas de Protozoários/química , Pirofosfatases/química , Antiprotozoários/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/enzimologia , Cristalografia por Raios X , Nucleotídeos de Desoxiuracil/metabolismo , Desoxiuridina/metabolismo , Desenho de Fármacos , Resistência a Medicamentos/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Pirofosfatases/antagonistas & inibidores , Pirofosfatases/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Biological macromolecules are polymers and therefore the restraints for macromolecular refinement can be subdivided into two sets: restraints that are applied to atoms that all belong to the same monomer and restraints that are associated with the covalent bonds between monomers. The CCP4 template-restraint library contains three types of data entries defining template restraints: descriptions of monomers and their modifications, both used for intramonomer restraints, and descriptions of links for intermonomer restraints. The library provides generic descriptions of modifications and links for protein, DNA and RNA chains, and for some post-translational modifications including glycosylation. Structure-specific template restraints can be defined in a user's additional restraint library. Here, JLigand, a new CCP4 graphical interface to LibCheck and REFMAC that has been developed to manage the user's library and generate new monomer entries is described, as well as new entries for links and associated modifications.
Assuntos
Bases de Dados de Proteínas , Design de Software , Algoritmos , Aminoácidos/química , Carboidratos/química , Ligantes , Modelos MolecularesRESUMO
We here aim to summarise the present knowledge on zinc binding by S100 proteins. While the importance of modulation of the function of the S100 family of EF-hand proteins by calcium is well established, a substantial proportion is also regulated by zinc or copper. Indeed regulation by zinc in addition to calcium was suggested almost as soon as the first S100 protein was discovered and has been confirmed for many family members by numerous experiments. For the first, "His-Zn", group, zinc-binding sites composed of three histidines and an aspartic acid were first proposed based on sequence comparisons and later confirmed by structural studies. A second, "Cys-Zn", group lacks such well-defined zinc-binding motifs and for these cysteines were suggested as the main zinc ligands. There is no three-dimensional structure for a Cys-Zn S100 in the presence of zinc. However, analysis of their sequences together with their X-ray structures in the absence of zinc suggests the possibility of two zinc-binding sites: a conserved site with a degree of similarity to those of the His-Zn group and a less-defined site with a Cys interdimer-binding motif. Some S100 protein-mediated events, such as signalling in the extracellular space, where the levels of calcium are already high, are most unlikely to be calcium regulated. Therefore, a broader knowledge of the role of zinc in the functioning of the S100 proteins will add significantly to the understanding how they propagate their signals.
Assuntos
Proteínas S100/química , Proteínas S100/metabolismo , Zinco/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Cisteína/química , Cisteína/metabolismo , Motivos EF Hand , Histidina/química , Histidina/metabolismo , Dados de Sequência Molecular , Zinco/químicaRESUMO
ß-Galactosidases catalyse the hydrolysis of lactose into galactose and glucose; as an alternative reaction, some ß-galactosidases also catalyse the formation of galactooligosaccharides by transglycosylation. Both reactions have industrial importance: lactose hydrolysis is used to produce lactose-free milk, while galactooligosaccharides have been shown to act as prebiotics. For some multi-domain ß-galactosidases, the hydrolysis/transglycosylation ratio can be modified by the truncation of carbohydrate-binding modules. Here, an analysis of BbgIII, a multidomain ß-galactosidase from Bifidobacterium bifidum, is presented. The X-ray structure has been determined of an intact protein corresponding to a gene construct of eight domains. The use of evolutionary covariance-based predictions made sequence docking in low-resolution areas of the model spectacularly easy, confirming the relevance of this rapidly developing deep-learning-based technique for model building. The structure revealed two alternative orientations of the CBM32 carbohydrate-binding module relative to the GH2 catalytic domain in the six crystallographically independent chains. In one orientation the CBM32 domain covers the entrance to the active site of the enzyme, while in the other orientation the active site is open, suggesting a possible mechanism for switching between the two activities of the enzyme, namely lactose hydrolysis and transgalactosylation. The location of the carbohydrate-binding site of the CBM32 domain on the opposite site of the module to where it comes into contact with the catalytic GH2 domain is consistent with its involvement in adherence to host cells. The role of the CBM32 domain in switching between hydrolysis and transglycosylation modes offers protein-engineering opportunities for selective ß-galactosidase modification for industrial purposes in the future.
Assuntos
Proteínas de Bactérias/metabolismo , Bifidobacterium bifidum/metabolismo , beta-Galactosidase/metabolismo , Proteínas de Bactérias/química , Bifidobacterium bifidum/enzimologia , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Galactose/metabolismo , Hidrólise , Lactose/metabolismo , Especificidade por Substrato , beta-Galactosidase/químicaRESUMO
Muramidases/lysozymes hydrolyse the peptidoglycan component of the bacterial cell wall. They are found in many of the glycoside hydrolase (GH) families. Family GH25 contains muramidases/lysozymes, known as CH type lysozymes, as they were initially discovered in the Chalaropsis species of fungus. The characterized enzymes from GH25 exhibit both ß-1,4-N-acetyl- and ß-1,4-N,6-O-diacetylmuramidase activities, cleaving the ß-1,4-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) moieties in the carbohydrate backbone of bacterial peptidoglycan. Here, a set of fungal GH25 muramidases were identified from a sequence search, cloned and expressed and screened for their ability to digest bacterial peptidoglycan, to be used in a commercial application in chicken feed. The screen identified the enzyme from Acremonium alcalophilum JCM 736 as a suitable candidate for this purpose and its relevant biochemical and biophysical and properties are described. We report the crystal structure of the A. alcalophilum enzyme at atomic, 0.78 Å resolution, together with that of its homologue from Trichobolus zukalii at 1.4 Å, and compare these with the structures of homologues. GH25 enzymes offer a new solution in animal feed applications such as for processing bacterial debris in the animal gut.
Assuntos
Fungos/metabolismo , Muramidase/metabolismo , Conformação Proteica , Animais , Parede Celular/metabolismo , Cristalografia por Raios X , Modelos MolecularesRESUMO
BACKGROUND: Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from Bacillus lentus by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site. However, the functional importance of such dynamics is unknown. METHODS: Here we have probed the conformational heterogeneity in Savinase by following the temperature dependent chemical shift changes. In addition, we have measured changes in the local stability of the enzyme when the inhibitor phenylmethylsulfonyl fluoride is bound using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Finally, we have used X-ray crystallography to compare electron densities collected at cryogenic and ambient temperatures and searched for possible low populated alternative conformations in the crystals. RESULTS: The NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified. The HDX shows that modification of Savinase with inhibitor has very little impact on the stability of hydrogen bonds and solvent accessibility of the backbone. The most pronounced structural heterogeneities detected in the diffraction data are limited to alternative side-chain rotamers and a short peptide segment that has an alternative main-chain conformation in the crystal at cryo conditions. Collectively, our data show that there is very little structural heterogeneity in the resting state of Savinase and hence that Savinase does not rely on conformational selection to drive the catalytic process.
RESUMO
BACKGROUND: Human S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins that are associated with many diseases including cancer, chronic inflammation and neurological disorders. S100A12 is an important factor in host/parasite defenses and in the inflammatory response. Like several other S100 proteins, it binds zinc and copper in addition to calcium. Mechanisms of zinc regulation have been proposed for a number of S100 proteins e.g. S100B, S100A2, S100A7, S100A8/9. The interaction of S100 proteins with their targets is strongly dependent on cellular microenvironment. RESULTS: The aim of the study was to explore the factors that influence S100A12 oligomerization and target interaction. A comprehensive series of biochemical and biophysical experiments indicated that changes in the concentration of calcium and zinc led to changes in the oligomeric state of S100A12. Surface plasmon resonance confirmed that the presence of both calcium and zinc is essential for the interaction of S100A12 with one of its extracellular targets, RAGE--the Receptor for Advanced Glycation End products. By using a single-molecule approach we have shown that the presence of zinc in tissue culture medium favors both the oligomerization of exogenous S100A12 protein and its interaction with targets on the cell surface. CONCLUSION: We have shown that oligomerization and target recognition by S100A12 is regulated by both zinc and calcium. Our present work highlighted the potential role of calcium-binding S100 proteins in zinc metabolism and, in particular, the role of S100A12 in the cross talk between zinc and calcium in cell signaling.
Assuntos
Cálcio/química , Multimerização Proteica , Proteínas S100/química , Zinco/química , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Cromatografia em Gel , Cristalografia por Raios X , Eletroforese em Gel Bidimensional , Humanos , Espectroscopia de Ressonância Magnética , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo , Proteínas S100/fisiologia , Proteína S100A12 , Transdução de Sinais , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Titulometria , Zinco/metabolismoRESUMO
Many proteins are synthesized as precursors, with propeptides playing a variety of roles such as assisting in folding or preventing them from being active within the cell. While the precise role of the propeptide in fungal lipases is not completely understood, it was previously reported that mutations in the propeptide region of the Rhizomucor miehei lipase have an influence on the activity of the mature enzyme, stressing the importance of the amino acid composition of this region. We here report two structures of this enzyme in complex with its propeptide, which suggests that the latter plays a role in the correct maturation of the enzyme. Most importantly, we demonstrate that the propeptide shows inhibition of lipase activity in standard lipase assays and propose that an important role of the propeptide is to ensure that the enzyme is not active during its expression pathway in the original host.
RESUMO
Endo-ß(1 â 4)-mannanases (endomannanases) catalyse degradation of ß-mannans, an abundant class of plant polysaccharides. This study investigates structural features and substrate binding of YpenMan26A, a non-CBM carrying endomannanase from Yunnania penicillata. Structural and sequence comparisons to other fungal family GH26 endomannanases showed high sequence similarities and conserved binding residues, indicating that fungal GH26 endomannanases accommodate galactopyranosyl units in the -3 and -2 subsites. Two striking amino acid differences in the active site were found when the YpenMan26A structure was compared to a homology model of Wsp.Man26A from Westerdykella sp. and the sequences of nine other fungal GH26 endomannanases. Two YpenMan26A mutants, W110H and D37T, inspired by differences observed in Wsp.Man26A, produced a shift in how mannopentaose bound across the active site cleft and a decreased affinity for galactose in the -2 subsite, respectively, compared to YpenMan26A. YpenMan26A was moreover found to have a flexible surface loop in the position where PansMan26A from Podospora anserina has an α-helix (α9) which interacts with its family 35 CBM. Sequence alignment inferred that the core structure of fungal GH26 endomannanases differ depending on the natural presence of this type of CBM. These new findings have implications for selecting and optimising these enzymes for galactomannandegradation.
Assuntos
Ascomicetos/enzimologia , Proteínas Fúngicas/química , Modelos Moleculares , Polissacarídeos/química , beta-Manosidase/química , Domínio Catalítico , Especificidade por SubstratoRESUMO
Glucoamylases are one of the most important classes of enzymes in the industrial degradation of starch biomass. They consist of a catalytic domain and a carbohydrate-binding domain (CBM), with the latter being important for the interaction with the polymeric substrate. Whereas the catalytic mechanisms and structures of the individual domains are well known, the spatial arrangement of the domains with respect to each other and its influence on activity are not fully understood. Here, the structures of three industrially used fungal glucoamylases, two of which are full length, have been crystallized and determined. It is shown for the first time that the relative orientation between the CBM and the catalytic domain is flexible, as they can adopt different orientations independently of ligand binding, suggesting a role as an anchor to increase the contact time and the relative concentration of substrate near the active site. The flexibility in the orientations of the two domains presented a considerable challenge for the crystallization of the enzymes.
Assuntos
Fungos/enzimologia , Glucana 1,4-alfa-Glucosidase/química , Sítios de Ligação , Metabolismo dos Carboidratos , Domínio Catalítico , Cristalização , Cristalografia/métodos , Proteínas Fúngicas/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Amido/metabolismoRESUMO
The enzymatic hydrolysis of complex plant biomass is a major societal goal of the 21st century in order to deliver renewable energy from nonpetroleum and nonfood sources. One of the major problems in many industrial processes, including the production of second-generation biofuels from lignocellulose, is the presence of `hemicelluloses' such as xylans which block access to the cellulosic biomass. Xylans, with a polymeric ß-1,4-xylose backbone, are frequently decorated with acetyl, glucuronyl and arabinofuranosyl `side-chain' substituents, all of which need to be removed for complete degradation of the xylan. As such, there is interest in side-chain-cleaving enzymes and their action on polymeric substrates. Here, the 1.25â Å resolution structure of the Talaromyces pinophilus arabinofuranosidase in complex with the inhibitor AraDNJ, which binds with a Kd of 24 ± 0.4â µM, is reported. Positively charged iminosugars are generally considered to be potent inhibitors of retaining glycosidases by virtue of their ability to interact with both acid/base and nucleophilic carboxylates. Here, AraDNJ shows good inhibition of an inverting enzyme, allowing further insight into the structural basis for arabinoxylan recognition and degradation.
Assuntos
Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Talaromyces/enzimologia , Cristalização , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
Bacteria use siderophores to mediate the transport of essential Fe(III) into the cell. In Campylobacter jejuni the periplasmic binding protein CeuE, an integral part of the Fe(III) transport system, has adapted to bind tetradentate siderophores using a His and a Tyr side chain to complete the Fe(III) coordination. A series of tetradentate siderophore mimics was synthesized in which the length of the linker between the two iron-binding catecholamide units was increased from four carbon atoms (4-LICAM4-) to five, six and eight (5-, 6-, 8-LICAM4-, respectively). Co-crystal structures with CeuE showed that the inter-planar angles between the iron-binding catecholamide units in the 5-, 6- and 8-LICAM4- structures are very similar (111°, 110° and 110°) and allow for an optimum fit into the binding pocket of CeuE, the inter-planar angle in the structure of 4-LICAM4- is significantly smaller (97°) due to restrictions imposed by the shorter linker. Accordingly, the protein-binding affinity was found to be slightly higher for 5- compared to 4-LICAM4- but decreases for 6- and 8-LICAM4-. The optimum linker length of five matches that present in natural siderophores such as enterobactin and azotochelin. Site-directed mutagenesis was used to investigate the relative importance of the Fe(III)-coordinating residues H227 and Y288.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Compostos Férricos/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Sideróforos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cristalografia por Raios X , Compostos Férricos/química , Regulação Bacteriana da Expressão Gênica , Proteínas de Ligação ao Ferro , Mutação , Periplasma/metabolismo , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genética , Ligação Proteica , Sideróforos/química , Espermidina/análogos & derivados , Espermidina/química , Espermidina/metabolismoRESUMO
Bacterial phosphoinositide-specific phospholipases C (PI-PLCs) are the smallest members of the PI-PLC family, which includes much larger mammalian enzymes responsible for signal transduction as well as enzymes from protozoan parasites, yeast and plants. Eukaryotic PI-PLCs have calcium in the active site, but this is absent in the known structures of Gram-positive bacteria, where its role is instead played by arginine. In addition to their use in a number of industrial applications, the bacterial enzymes attract special interest because they can serve as convenient models of the catalytic domains of eukaryotic enzymes for in vitro activity studies. Here, the structure of a PI-PLC from Pseudomonas sp. 62186 is reported, the first from a Gram-negative bacterium and the first of a native bacterial PI-PLC with calcium present in the active site. Solution of the structure posed particular problems owing to the low sequence identity of available homologous structures. Its dependence on calcium for catalysis makes this enzyme a better model for studies of the mammalian PI-PLCs than the previously used calcium-independent bacterial PI-PLCs.