RESUMO
Transthyretin (TTR) is a homo-tetrameric serum protein associated with sporadic and hereditary systemic amyloidosis. TTR amyloid formation proceeds by the dissociation of the TTR tetramer and the subsequent partial unfolding of the TTR monomer into an aggregation-prone conformation. Although TTR kinetic stabilizers suppress tetramer dissociation, a strategy for stabilizing monomers has not yet been developed. Here, we show that an N-terminal C10S mutation increases the thermodynamic stability of the TTR monomer by forming new hydrogen bond networks through the side chain hydroxyl group of Ser10. Nuclear magnetic resonance spectrometry and molecular dynamics simulation revealed that the Ser10 hydroxyl group forms hydrogen bonds with the main chain amide group of either Gly57 or Thr59 on the DE loop. These hydrogen bonds prevent the dissociation of edge strands in the DAGH and CBEF ß-sheets during the unfolding of the TTR monomer by stabilizing the interaction between ß-strands A and D and the quasi-helical structure in the DE loop. We propose that introducing hydrogen bonds to connect the N-terminal region to the DE loop reduces the amyloidogenic potential of TTR by stabilizing the monomer.
Assuntos
Simulação de Dinâmica Molecular , Pré-Albumina , Conformação Proteica , Ligação de Hidrogênio , Pré-Albumina/química , Pré-Albumina/genética , Pré-Albumina/metabolismo , Amiloide/química , Amiloide/metabolismoRESUMO
Fragment-based drug discovery (FBDD), which involves small compounds <300 Da, has been recognized as one of the most powerful tools for drug discovery. In FBDD, the affinity of hit compounds tends to be low, and the analysis of protein-compound interactions becomes difficult. In an effort to overcome such difficulty, we developed a 19F-NMR screening method optimizing a 19F chemical library focusing on highly soluble monomeric molecules. Our method was successfully applied to four proteins, including protein kinases and a membrane protein. For FKBP12, hit compounds were carefully validated by protein thermal shift analysis, 1H-15N HSQC NMR spectroscopy, and isothermal titration calorimetry to determine dissociation constants and model complex structures. It should be noted that the 1H and 19F saturation transfer difference experiments were crucial to obtaining highly precise model structures. The combination of 19F-NMR analysis and the optimized 19F chemical library enables the modeling of the complex structure made up of a weak binder and its target protein.
RESUMO
Structure-based high-throughput screening of chemical compounds that target protein-protein interactions (PPIs) is a promising technology for gaining insight into how plant development is regulated, leading to many potential agricultural applications. At present, there are no examples of using high-throughput screening to identify chemicals that target plant transcriptional complexes, some of which are responsible for regulating multiple physiological functions. Florigen, a protein encoded by FLOWERING LOCUS T (FT), was initially identified as a molecule that promotes flowering and has since been shown to regulate flowering and other developmental phenomena such as tuber formation in potato (Solanum tuberosum). FT functions as a component of the florigen activation complex (FAC) with a 14-3-3 scaffold protein and FD, a bZIP transcription factor that activates downstream gene expression. Although 14-3-3 is an important component of FAC, little is known about the function of the 14-3-3 protein itself. Here, we report the results of a high-throughput in vitro fluorescence resonance energy transfer (FRET) screening of chemical libraries that enabled us to identify small molecules capable of inhibiting FAC formation. These molecules abrogate the in vitro interaction between the 14-3-3 protein and the OsFD1 peptide, a rice (Oryza sativa) FD, by directly binding to the 14-3-3 protein. Treatment with S4, a specific hit molecule, strongly inhibited FAC activity and flowering in duckweed, tuber formation in potato, and branching in rice in a dose-dependent manner. Our results demonstrate that the high-throughput screening approach based on the three-dimensional structure of PPIs is suitable in plants. In this study, we have proposed good candidate compounds for future modification to obtain inhibitors of florigen-dependent processes through inhibition of FAC formation.
Assuntos
Florígeno , Oryza , Florígeno/metabolismo , Proteínas de Plantas/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Ensaios de Triagem em Larga Escala , Oryza/metabolismo , Regulação da Expressão Gênica de Plantas , Flores/genéticaRESUMO
GH19 and GH22 glycoside hydrolases belonging to the lysozyme superfamily have a related structure/function. A highly conserved tryptophan residue, Trp103, located in the binding groove of a GH19 chitinase from moss Bryum coronatum (BcChi-A) appears to have a function similar to that of well-known Trp62 in GH22 lysozymes. Here, we found that mutation of Trp103 to phenylalanine (W103F) or alanine (W103A) strongly reduced the enzymatic activity of BcChi-A. NMR experiments and the X-ray crystal structure suggested a hydrogen bond between the Trp103 side chain and the -2 sugar. Chitooligosaccharide binding experiments using NMR indicated that the W103F mutation reduced the sugar-binding abilities of nearby amino acid residues (Tyr105/Asn106) in addition to Trp103. This appeared to be derived from enhanced aromatic stacking of Phe103 with Tyr105 induced by disruption of the Trp103 hydrogen bond with the -2 sugar. Since the stacking with Tyr105 was unlikely in W103A, Tyr105/Asn106 of W103A was not so affected as in W103F. However, the W103A mutation appeared to reduce the catalytic potency, resulting in the lowest enzymatic activity in W103A. We concluded that Trp103 does not only interact with the sugar, but also controls other amino acids responsible for substrate binding and catalysis. Trp103 (GH19) and Trp62 (GH22) with such a multi-functionality may be advantageous for enzyme action and conserved in the divergent evolution in the lysozyme superfamily.
Assuntos
Bryopsida/enzimologia , Quitina/análogos & derivados , Quitinases/química , Proteínas de Plantas/química , Substituição de Aminoácidos , Sítios de Ligação , Bryopsida/genética , Quitina/química , Quitinases/genética , Quitosana , Mutação de Sentido Incorreto , Oligossacarídeos , Proteínas de Plantas/genética , Triptofano/química , Triptofano/genéticaRESUMO
BACKGROUND: In protein crystals, flexible loops are frequently deformed by crystal contacts, whereas in solution, the large motions result in the poor convergence of such flexible loops in NMR structure determinations. We need an experimental technique to characterize the structural and dynamic properties of intrinsically flexible loops of protein molecules. METHODS: We designed an intended crystal contact-free space (CCFS) in protein crystals, and arranged the flexible loop of interest in the CCFS. The yeast Tim 21 protein was chosen as the model protein, because one of the loops (loop 2) is distorted by crystal contacts in the conventional crystal. RESULTS: Yeast Tim21 was fused to the MBP protein by a rigid α-helical linker. The space created between the two proteins was used as the CCFS. The linker length provides adjustable freedom to arrange loop 2 in the CCFS. We re-determined the NMR structure of yeast Tim21, and conducted MD simulations for comparison. Multidimensional scaling was used to visualize the conformational similarity of loop 2. We found that the crystal contact-free conformation of loop 2 is located close to the center of the ensembles of the loop 2 conformations in the NMR and MD structures. CONCLUSIONS: Loop 2 of yeast Tim21 in the CCFS adopts a representative, dominant conformation in solution. GENERAL SIGNIFICANCE: No single powerful technique is available for the characterization of flexible structures in protein molecules. NMR analyses and MD simulations provide useful, but incomplete information. CCFS crystallography offers a third route to this goal.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli , Espectroscopia de Ressonância Magnética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Simulação de Dinâmica Molecular , Movimento (Física) , Estrutura Secundária de Proteína , Eletricidade EstáticaRESUMO
Motivation: Multi-dimensional NMR spectra are generally used for NMR signal assignment and structure analysis. There are several programs that can achieve highly automated NMR signal assignments and structure analysis. On the other hand, NMR spectra tend to have a large number of noise peaks even for data acquired with good sample and machine conditions, and it is still difficult to eliminate these noise peaks. Results: We have developed a method to eliminate noise peaks using convolutional neural networks, implemented in the program package Filt_Robot. The filtering accuracy of Filt_Robot was around 90-95% when applied to 2D and 3D NMR spectra, and the numbers of resulting non-noise peaks were close to those in corresponding manually prepared peaks lists. The filtering can strongly enhance automated NMR spectra analysis. Availability and implementation: The full package of the program, documents and example data are available from http://bmrbdep.pdbj.org/en/nmr_tool_box/Filt_Robot.html. Supplementary information: Supplementary data are available at Bioinformatics online.
Assuntos
Espectroscopia de Ressonância Magnética , Redes Neurais de Computação , Proteínas , SoftwareRESUMO
Due to their lower production cost compared with monoclonal antibodies, single-chain variable fragments (scFvs) have potential for use in several applications, such as for diagnosis and treatment of a range of diseases, and as sensor elements. However, the usefulness of scFvs is limited by inhomogeneity through the formation of dimers, trimers, and larger oligomers. The scFv protein is assumed to be in equilibrium between the closed and open states formed by assembly or disassembly of VH and VL domains. Therefore, the production of an scFv with equilibrium biased to the closed state would be critical to overcome the problem in inhomogeneity of scFv for industrial or therapeutic applications. In this study, we obtained scFv clones stable against GA-pyridine, an advanced glycation end-product (AGE), by using a combination of a phage display system and random mutagenesis. Executing the bio-panning at 37 °C markedly improved the stability of scFvs. We further evaluated the radius of gyration by small-angle X-ray scattering (SAXS), obtained compact clones, and also visualized open.
Assuntos
Produtos Finais de Glicação Avançada/imunologia , Compostos de Piridínio/imunologia , Anticorpos de Cadeia Única/biossíntese , Sequência de Aminoácidos , Biblioteca de Peptídeos , Domínios Proteicos , Multimerização Proteica , Estabilidade Proteica , Anticorpos de Cadeia Única/químicaRESUMO
We review studies on biochemical characterization of the structures and functions of chitinase, chitosanase, and chitobiase produced by cells of the bacterium, Paenibacillus sp. IK-5. The IK-5 chitinases comprise two GH18 chitinases (ChiA and ChiB), an auxiliary activity family 10 (AA10) chitin oxydehydrolase (ChiC), and a GH19 chitinase (ChiD). The IK-5 chitosanase (ChiE) has a glycosyl hydrolase family 8 (GH8) catalytic domain at the amino-terminus and two discoidin domains (DD) at the carboxyl-terminus. The IK-5 cells also produce chitobiase, containing carbohydrate hydrolase H-20 and S-layer homology domains. Together, these ChiAâ¼ChiE proteins form a huge complex, designated the "chitinasome". The DD domains bind specifically and tightly to chitosan, suggesting that they are chitosan-specific carbohydrate-binding modules (CBM32); indeed, CBM32 modules have been confirmed to bind to chitosan oligosaccharides (GlcN)2-6. A high-yield secretion system for Ik-5 chitosanase has been constructed using plasmid pNY301 expressed in Bacillus brevis. We also review biotechnological research using chitin, chitosan, crab shell, and IK-5 chitinase and chitosanase. Chitosan has been shown to be useful for efficient gene transfer into microbial and animal cells. IK-5 cell culture and crab shells were effective for the growth of plants and seaweeds.
Assuntos
Biotecnologia/métodos , Quitina/biossíntese , Quitinases/biossíntese , Quitosana/metabolismo , Glicosídeo Hidrolases/biossíntese , Paenibacillus/metabolismo , Quitina/química , Quitina/farmacologia , Quitosana/química , Quitosana/farmacologiaRESUMO
Chitosan interaction with chitosanase was examined through analysis of spectral line shapes in the NMR HSQC titration experiments. We established that the substrate, chitosan hexamer, binds to the enzyme through the three-state induced-fit mechanism with fast formation of the encounter complex followed by slow isomerization of the bound-state into the final conformation. Mapping of the chemical shift perturbations in two sequential steps of the mechanism highlighted involvement of the substrate-binding subsites and the hinge region in the binding reaction. Equilibrium parameters of the three-state model agreed with the overall thermodynamic dissociation constant determined by ITC. This study presented the first kinetic evidence of the induced-fit mechanism in the glycoside hydrolases.
Assuntos
Glicosídeo Hidrolases/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Quitosana/química , Quitosana/metabolismo , Glicosídeo Hidrolases/química , Cinética , Ligantes , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Chitosan-related enzymes including chitosanases, exo-ß-glucosaminidases, and enzymes having chitosan-binding modules recognize ligands through electrostatic interactions between the acidic amino acids in proteins and amino groups of chitosan polysaccharides. However, in GH8 chitosanases, several aromatic residues are also involved in substrate recognition through stacking interactions, and these enzymes consequently hydrolyze ß-1,4-glucan as well as chitosan. The binding grooves of these chitosanases are extended and opened at both ends of the grooves, so that the enzymes can clamp a long chitosan polysaccharide. The association/dissociation of positively charged glucosamine residues to/from the binding pocket of a GH2 exo-ß-glucosaminidase controls the p Ka of the catalytic acid, thereby maintaining the high catalytic potency of the enzyme. In contrast to chitosanases, chitosan-binding modules only accommodate a couple of glucosamine residues, predominantly recognizing the non-reducing end glucosamine residue of chitosan by electrostatic interactions and a hydrogen-bonding network. These structural findings on chitosan-related enzymes may contribute to future applications for the efficient conversion of the chitin/chitosan biomass.
Assuntos
Quitosana/metabolismo , Glicosídeo Hidrolases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Glicosídeo Hidrolases/química , Ligação ProteicaRESUMO
Two-component signal transduction systems (TCSs), composed of a histidine kinase sensor (HK) and its cognate response regulator, sense and respond to environmental changes and are related to the virulence of pathogens. TCSs are potential targets for alternative antibiotics and anti-virulence agents. Here we found that waldiomycin, an angucycline antibiotic that inhibits a growth essential HK, WalK, in Gram-positive bacteria, also inhibits several class I HKs from the Gram-negative Escherichia coli. NMR analyses and site-directed mutagenesis studies using the osmo-sensing EnvZ, a prototypical HK of E. coli, showed that waldiomycin directly binds to both H-box and X-region, which are the two conserved regions in the dimerization-inducing and histidine-containing phosphotransfer (DHp) domain of HKs. Waldiomycin inhibits phosphorylation of the conserved histidine in the H-box. Analysis of waldiomycin derivatives suggests that the angucyclic ring, situated near the H-box in the waldiomycin-EnvZ DHp domain complex model, is responsible for the inhibitory activity. We demonstrate that waldiomycin is an HK inhibitor binding to the H-box region and has the potential of inhibiting a broad spectrum of HKs.
Assuntos
Antibacterianos/farmacologia , Histidina Quinase/antagonistas & inibidores , Histidina Quinase/química , Quinonas/farmacologia , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas da Membrana Bacteriana Externa/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/genética , Sequência Conservada , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Histidina Quinase/genética , Modelos Estruturais , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/efeitos dos fármacos , Complexos Multienzimáticos/genética , Mutagênese Sítio-Dirigida , FosforilaçãoRESUMO
An antifungal chitosanase/glucanase isolated from the soil bacterium Paenibacillus sp. IK-5 has two CBM32 chitosan-binding modules (DD1 and DD2) linked in tandem at the C-terminus. In order to obtain insights into the mechanism of chitosan recognition, the structures of DD1 and DD2 were solved by NMR spectroscopy and crystallography. DD1 and DD2 both adopted a ß-sandwich fold with several loops in solution as well as in crystals. On the basis of chemical shift perturbations in(1)H-(15)N-HSQC resonances, the chitosan tetramer (GlcN)4 was found to bind to the loop region extruded from the core ß-sandwich of DD1 and DD2. The binding site defined by NMR in solution was consistent with the crystal structure of DD2 in complex with (GlcN)3, in which the bound (GlcN)3 stood upright on its non-reducing end at the binding site. Glu(14)of DD2 appeared to make an electrostatic interaction with the amino group of the non-reducing end GlcN, and Arg(31), Tyr(36)and Glu(61)formed several hydrogen bonds predominantly with the non-reducing end GlcN. No interaction was detected with the reducing end GlcN. Since Tyr(36)of DD2 is replaced by glutamic acid in DD1, the mutation of Tyr(36)to glutamic acid was conducted in DD2 (DD2-Y36E), and the reverse mutation was conducted in DD1 (DD1-E36Y). Ligand-binding experiments using the mutant proteins revealed that this substitution of the 36th amino acid differentiates the binding properties of DD1 and DD2, probably enhancing total affinity of the chitosanase/glucanase toward the fungal cell wall.
Assuntos
Proteínas de Bactérias/metabolismo , Quitosana/metabolismo , Glicosídeo Hidrolases/metabolismo , Paenibacillus , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/fisiologia , Quitosana/química , Cristalografia por Raios X , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Especificidade por Substrato/fisiologiaRESUMO
A goose-type lysozyme from ostrich egg white (OEL) was produced by Escherichia coli expression system, and the role of His101 of OEL in the enzymatic reaction was investigated by NMR spectroscopy, thermal unfolding, and theoretical modeling of the enzymatic hydrolysis of hexa-N-acetylchitohexaose, (GlcNAc)6. Although the binding of tri-N-acetylchitotriose, (GlcNAc)3, to OEL perturbed several backbone resonances in the (1)H-(15)N HSQC spectrum, the chemical shift of the backbone resonance of His101 was not significantly affected. However, apparent pKa values of His101 and Lys102 determined from the pH titration curves of the backbone chemical shifts were markedly shifted by (GlcNAc)3 binding. Thermal unfolding experiments and modeling study of (GlcNAc)6 hydrolysis using a His101-mutated OEL (H101A-OEL) revealed that the His101 mutation affected not only sugar residue affinities at subsites -3 and -2 but also the rate constant for bond cleavage. His101 appears to play multiple roles in the substrate binding and the catalytic reaction.
Assuntos
Proteínas Aviárias/química , Histidina/química , Muramidase/química , Oligossacarídeos/química , Trissacarídeos/química , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Sítios de Ligação , Clonagem Molecular , Clara de Ovo/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Modelos Moleculares , Muramidase/genética , Muramidase/metabolismo , Ressonância Magnética Nuclear Biomolecular , Oligossacarídeos/metabolismo , Ligação Proteica , Desdobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Struthioniformes , Especificidade por Substrato , Trissacarídeos/metabolismo , Zigoto/químicaRESUMO
Enhancing the transglycosylation (TG) activity of glycoside hydrolases does not always result in the production of oligosaccharides with longer chains, because the TG products are often decomposed into shorter oligosaccharides. Here, we investigated the mutation strategies for obtaining chitooligosaccharides with longer chains by means of TG reaction catalyzed by family GH18 chitinase A from Vibrio harveyi (VhChiA). HPLC analysis of the TG products from incubation of chitooligosaccharide substrates, GlcNAc(n), with several mutant VhChiAs suggested that mutant W570G (mutation of Trp570 to Gly) and mutant D392N (mutation of Asp392 to Asn) significantly enhanced TG activity, but the TG products were immediately hydrolyzed into shorter GlcNAc(n). On the other hand, the TG products obtained from mutants D313A and D313N (mutations of Asp313 to Ala and Asn, respectively) were not further hydrolyzed, leading to the accumulation of oligosaccharides with longer chains. The data obtained from the mutant VhChiAs suggested that mutations of Asp313, the middle aspartic acid residue of the DxDxE catalytic motif, to Ala and Asn are most effective for obtaining chitooligosaccharides with longer chains.
Assuntos
Proteínas de Bactérias/genética , Quitinases/genética , Mutação , Oligossacarídeos/biossíntese , Vibrio/genética , Acetilglucosamina/metabolismo , Alanina/química , Alanina/metabolismo , Asparagina/química , Asparagina/metabolismo , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Quitinases/química , Quitinases/metabolismo , Expressão Gênica , Glicosilação , Hidrólise , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Engenharia de Proteínas , Vibrio/enzimologiaRESUMO
Tri-N-acetylchitotriosyl moranoline, (GlcNAc)3-M, was previously shown to strongly inhibit lysozyme (Ogata M, Umemoto N, Ohnuma T, Numata T, Suzuki A, Usui T, Fukamizo T. 2013. A novel transition-state analogue for lysozyme, 4-O-ß-tri-Nacetylchitotriosyl moranoline, provided evidence supporting the covalent glycosyl-enzyme intermediate. J Biol Chem. 288:6072-6082). The findings prompted us to examine the interaction of di-N-acetylchitobiosyl moranoline, (GlcNAc)2-M, with a family GH19 chitinase from moss, Bryum coronatum (BcChi19A). Thermal unfolding experiments using BcChi19A and the catalytic acid-deficient mutant (BcChi19A-E61A) revealed that the transition temperature (Tm) was elevated by 4.3 and 5.8°C, respectively, upon the addition of (GlcNAc)2-M, while the chitin dimer, (GlcNAc)2, elevated Tm only by 1.0 and 1.4°C, respectively. By means of isothermal titration calorimetry, binding free energy changes for the interactions of (GlcNAc)3 and (GlcNAc)2-M with BcChi19A-E61A were determined to be -5.2 and -6.6 kcal/mol, respectively, while (GlcNAc)2 was found to interact with BcChi19A-E61A with markedly lower affinity. nuclear magnetic resonance titration experiments using (15)N-labeled BcChi19A and BcChi19A-E61A revealed that both (GlcNAc)2 and (GlcNAc)2-M interact with the region surrounding the catalytic center of the enzyme and that the interaction of (GlcNAc)2-M is markedly stronger than that of (GlcNAc)2 for both enzymes. However, (GlcNAc)2-M was found to moderately inhibit the hydrolytic reaction of chitin oligosaccharides catalyzed by BcChi19A (IC50 = 130-620 µM). A molecular dynamics simulation of BcChi19A in complex with (GlcNAc)2-M revealed that the complex is quite stable and the binding mode does not significantly change during the simulation. The moranoline moiety of (GlcNAc)2-M did not fit into the catalytic cleft (subsite -1) but was rather in contact with subsite +1. This situation may result in the moderate inhibition toward the BcChi19A-catalyzed hydrolysis.
Assuntos
1-Desoxinojirimicina/metabolismo , Quitinases/metabolismo , Dissacarídeos/metabolismo , 1-Desoxinojirimicina/química , Calorimetria , Domínio Catalítico , Quitina/química , Quitina/metabolismo , Quitinases/química , Dissacarídeos/química , Hidrólise , Espectroscopia de Ressonância Magnética , Muramidase/antagonistas & inibidores , Muramidase/química , Ligação Proteica , Sphagnopsida/químicaRESUMO
DESCRIPTIONS: The structure of a GH19 chitinase from the moss Bryum coronatum (BcChi-A) in complex with the substrate was examined by X-ray crystallography and NMR spectroscopy in solution. The X-ray crystal structure of the inactive mutant of BcChi-A (BcChi-A-E61A) liganded with chitin tetramer (GlcNAc)4 revealed a clear electron density of the tetramer bound to subsites -2, -1, +1, and +2. Individual sugar residues were recognized by several amino acids at these subsites through a number of hydrogen bonds. This is the first crystal structure of GH19 chitinase liganded with oligosaccharide spanning the catalytic center. NMR titration experiments of chitin oligosaccharides into the BcChi-A-E61A solution showed that the binding mode observed in the crystal structure is similar to that in solution. The C-1 carbon of -1 GlcNAc, the Oε1 atom of the catalytic base (Glu70), and the Oγ atom of Ser102 form a "triangle" surrounding the catalytic water, and the arrangement structurally validated the proposed catalytic mechanism of GH19 chitinases. The glycosidic linkage between -1 and +1 sugars was found to be twisted and under strain. This situation may contribute to the reduction of activation energy for hydrolysis. The complex structure revealed a more refined mechanism of the chitinase catalysis.
Assuntos
Bryopsida/química , Quitina/química , Quitinases/química , Modelos Moleculares , Proteínas de Plantas/química , Bryopsida/enzimologia , Bryopsida/genética , Domínio Catalítico , Quitina/metabolismo , Quitinases/genética , Quitinases/metabolismo , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Ligação de Hidrogênio , Hidrólise , Espectroscopia de Ressonância Magnética , Mutação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
The intact cells of Rhizopus oligosporus NRRL2710, whose cell walls are abundant source of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN), were digested with three chitinolytic enzymes, a GH-46 chitosanase from Streptomyces sp. N174 (CsnN174), a chitinase from Pyrococcus furiosus, and a chitinase from Trichoderma viride, respectively. Solubilization of the intact cells by CsnN174 was found to be the most efficient from solid state CP/MAS (13)C NMR spectroscopy. Chitosanase products from Rhizopus cells were purified by cation exchange chromatography on CM-Sephadex C-25 and gel-filtration on Cellulofine Gcl-25m. NMR and MALDI-TOF-MS analyses of the purified products revealed that GlcN-GlcNAc, (GlcN)2-GlcNAc, and (GlcN)2 were produced by the enzymatic digestion of the intact cells. The chitosanase digestion of Rhizopus cells was found to be an excellent system for the conversion of fungal biomass without any environmental impact.
Assuntos
Acetilglucosamina/isolamento & purificação , Glucosamina/química , Glicosídeo Hidrolases/metabolismo , Rhizopus/química , Acetilglucosamina/química , Parede Celular/química , Quitosana/química , Cromatografia Líquida de Alta Pressão , Glucosamina/isolamento & purificação , Espectroscopia de Ressonância Magnética , Pyrococcus/enzimologia , Rhizopus/metabolismo , Streptomyces/enzimologia , Trichoderma/enzimologiaRESUMO
Two carbohydrate binding modules (DD1 and DD2) belonging to CBM32 are located at the C terminus of a chitosanase from Paenibacillus sp. IK-5. We produced three proteins, DD1, DD2, and tandem DD1/DD2 (DD1+DD2), and characterized their binding ability. Transition temperature of thermal unfolding (Tm) of each protein was elevated by the addition of cello-, laminari-, chitin-, or chitosan-hexamer (GlcN)6. The Tm elevation (ΔTm) in DD1 was the highest (10.3 °C) upon the addition of (GlcN)6 and was markedly higher than that in DD2 (1.0 °C). A synergistic effect was observed (ΔTm = 13.6 °C), when (GlcN)6 was added to DD1+DD2. From isothermal titration calorimetry experiments, affinities to DD1 were not clearly dependent upon chain length of (GlcN)n; ΔGr° values were -7.8 (n = 6), -7.6 (n = 5), -7.6 (n = 4), -7.6 (n = 3), and -7.1 (n = 2) kcal/mol, and the value was not obtained for GlcN due to the lowest affinity. DD2 bound (GlcN)n with the lower affinities (ΔGr° = -5.0 (n = 3) ~ -5.2 (n = 6) kcal/mol). Isothermal titration calorimetry profiles obtained for DD1+DD2 exhibited a better fit when the two-site model was used for analysis and provided greater affinities to (GlcN)6 for individual DD1 and DD2 sites (ΔGr° = -8.6 and -6.4 kcal/mol, respectively). From NMR titration experiments, (GlcN)n (n = 2~6) were found to bind to loops extruded from the core ß-sandwich of individual DD1 and DD2, and the interaction sites were similar to each other. Taken together, DD1+DD2 is specific to chitosan, and individual modules synergistically interact with at least two GlcN units, facilitating chitosan hydrolysis.
Assuntos
Proteínas de Bactérias/química , Quitosana/química , Glicosídeo Hidrolases/química , Paenibacillus/enzimologia , Desdobramento de Proteína , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Quitosana/metabolismo , Estabilidade Enzimática , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Chitinase C from Ralstonia sp. A-471 (Ra-ChiC) has a catalytic domain sequence similar to goose-type (G-type) lysozymes and, unlike other chitinases, belongs to glycohydrolase (GH) family 23. Using NMR spectroscopy, however, Ra-ChiC was found to interact only with the chitin dimer but not with the peptidoglycan fragment. Here we report the crystal structures of wild-type, E141Q, and E162Q of the catalytic domain of Ra-ChiC with or without chitin oligosaccharides. Ra-ChiC has a substrate-binding site including a tunnel-shaped cavity, which determines the substrate specificity. Mutation analyses based on this structural information indicated that a highly conserved Glu-141 acts as a catalytic acid, and that Asp-226 located at the roof of the tunnel activates a water molecule as a catalytic base. The unique arrangement of the catalytic residues makes a clear contrast to the other GH23 members and also to inverting GH19 chitinases.