RESUMEN
We have investigated the mechanism of the interaction of Streptomyces sp. N174 chitosanase with glucosamine hexasaccharide [(GlcN)(6)] by site-directed mutagenesis, thermal unfolding, and (GlcN)(6) digestion experiments, followed by theoretical calculations. From the energy-minimized model of the chitosanase-(GlcN)(6) complex structure (Marcotte et al., 1996), Asp57, which is present in all known chitosanases, was proposed to be one of the amino acid residues that interacts with the oligosaccharide substrate. The chitosanase gene was mutated at Asp57 to Asn (D57N) and Ala (D57A), and the relative activities of the mutated chitosanases were found to be 72 and 0.5% of that of the wild type, respectively. The increase in the transition temperature of thermal unfolding (T(m)), usually observed upon the addition of (GlcN)(n) to chitosanase mutants unaffected in terms of substrate binding, was considerably suppressed in the D57A mutant. These data suggest that Asp57 is important for substrate binding. The experimental time-courses of [(GlcN)(6)] degradation were analyzed by a theoretical model in order to obtain the binding free energy values of the individual subsites of the chitosanases. A (-3, -2, -1, +1, +2, +3) subsite model agreed best with the experimental data. This analysis also indicated that the mutation of Asp57 affects substrate affinity at subsite (-2), suggesting that Asp57 most likely participates in the substrate binding at this subsite.
Asunto(s)
Quitina/metabolismo , Glicósido Hidrolasas/metabolismo , Oligosacáridos/metabolismo , Streptomyces/enzimología , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Secuencia de Aminoácidos , Ácido Aspártico/metabolismo , Sitios de Unión/fisiología , Quitina/análogos & derivados , Quitina/química , Quitinasas/metabolismo , Quitosano , Secuencia Conservada , Glucosamina/química , Glucosamina/metabolismo , Hordeum/enzimología , Calor , Cinética , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Pliegue de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Streptomyces/metabolismo , Termodinámica , Virus/enzimologíaRESUMEN
Binding of a highly de-N-acetylated chitosan to Japanese pheasant lysozyme (JPL), which differs from hen egg white lysozyme (HEWL) by nine amino acid substitutions (including Arg114-->His), was investigated by 1H-NMR spectroscopy. The profile of the one-dimensional spectrum of JPL is essentially identical to that of HEWL. Using two-dimensional spectra of JPL and HEWL, several aromatic and aliphatic proton resonances of JPL were assigned by comparison. When a highly de-N-acetylated chitosan (number-average degree of polymerization, about 18; degree of acetylation, 0.04), where the N-acetylated units are predominantly surrounded by de-N-acetylated units (a monoacetylated chitosan), was added to the JPL solution, the NMR signals were clearly affected in Trp28 C5H and Ile98 gammaCH, as in the case of binding to HEWL. The dissociation constant of the monoacetylated chitosan evaluated from the NMR signal responses was calculated to be 0.23+/-0.05 mm (-31.5 kJ/mol), which is similar to that of HEWL (0.11+/-0.02 mm, -33.3 kJ/mol). Thus, the Arg-->His substitution of the 114th amino acid, which participates in sugar residue binding at the right-sided subsite F, did not significantly affect the chitosan binding. In addition, the C2H signal of His114 of JPL was not affected by the chitosan binding. These results suggest that the monoacetylated chitosan binds to subsites E and F through the left-sided binding mode.
Asunto(s)
Aves/metabolismo , Quitina/química , Muramidasa/química , Algoritmos , Secuencia de Aminoácidos , Animales , Quitina/análogos & derivados , Quitosano , Remoción de Radical Alquila , Óxido de Deuterio , Clara de Huevo/análisis , Electroquímica , Concentración de Iones de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Unión Proteica , Triptófano/químicaRESUMEN
Manduca sexta (tobacco hornworm) chitinase is a molting enzyme that contains several domains including a catalytic domain, a serine/threonine-rich region, and a C-terminal cysteine-rich domain. Previously we showed that this chitinase acts as a biopesticide in transgenic plants where it disrupts gut physiology. To delineate the role of these domains further and to identify and characterize some of the multiple forms produced in molting fluid and in transgenic plants, three different forms with variable lengths of C-terminal deletions were generated. Appropriately truncated forms of the M. sexta chitinase cDNA were generated, introduced into a baculovirus vector, and expressed in insect cells. Two of the truncated chitinases (Chi 1-407 and Chi 1-477) were secreted into the medium, whereas the one with the longest deletion (Chi 1-376) was retained inside the insect cells. The two larger truncated chitinases and the full-length enzyme (Chi 1-535) were purified and their properties were compared. Differences in carbohydrate compositions, pH-activity profiles, and kinetic constants were observed among the different forms of chitinases. All three of these chitinases had some affinity for chitin, and they also exhibited differences in their ability to hydrolyze colloidal chitin. The results support the hypothesis that multiple forms of this enzyme occur in vivo due to proteolytic processing at the C-terminal end and differential glycosylation.
Asunto(s)
Quitinasas/metabolismo , Manduca/enzimología , Animales , Quitina/metabolismo , Quitinasas/genética , Glicosilación , Concentración de Iones de Hidrógeno , Cinética , Mutagénesis , Unión Proteica , Procesamiento Proteico-Postraduccional , Señales de Clasificación de Proteína , TemperaturaRESUMEN
The endochitinase from Coccidioides immitis (CiX1) is a member of the class 18 chitinase family. Here we show the enzyme functions by a retaining catalytic mechanism; that is, the beta-conformation of the chitin substrate linkages is preserved after hydrolysis. The pattern of cleavage of N-acetyglucosamine (GlcNAc) oligosaccharide substrates has been determined. (GlcNAc)6 is predominantly cleaved into (GlcNAc)2 and (GlcNAc)4, where the (GlcNAc)2 group arises from the nonreducing end of the substrate and is formed as the beta-anomer. With time, transglycosylation occurs, generating (GlcNAc)8 from the product dimer and fresh hexamer. Similar patterns are seen for the cleavage of (GlcNAc)5 and (GlcNAc)4 where dimers cleaved from the nonreducing end reflect the most common binding and hydrolysis pattern. Intrinsic fluorescence measurements suggest the dissociation constant for (GlcNAc)4 is 50 microM. Synthetic substrates with fluorescent leaving groups exhibit complicated profiles in the relationship between initial velocity and substrate concentration, making it difficult to obtain the values of kinetic constants. An improved theoretical analysis of the time-course of (GlcNAc)6 degradation allows the unitary free energy of binding of the individual subsites of the enzyme to be estimated. The free energy values obtained are consistent with the dissociation constant obtained by fluorescence measurements, and generate a model of substrate interaction that can be tested against the crystal structure of the enzyme.
Asunto(s)
Quitinasas/metabolismo , Coccidioides/enzimología , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Acetilglucosamina/metabolismo , Sitios de Unión , Coccidioides/patogenicidad , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Oligosacáridos/metabolismo , Especificidad por Sustrato , Factores de TiempoRESUMEN
The kinetic behavior of chitinase A1 from Bacillus circulans WL-12 was investigated using the novel fluorogenic substrates, N-deacetylated 4-methylumbelliferyl chitobiosides [GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)(2)-UMB (4)], and the results were compared with those obtained using 4-methylumbelliferyl N, N'-diacetylchitobiose [(GlcNAc)(2)-UMB (1)] as the substrate. The chitinase did not release the UMB moiety from compound 4, but successfully released UMB from the other substrates. k(cat)/K(m) values determined from the releasing rate of the UMB moiety were: 145.3 for 1, 8.3 for 2, and 0.1 s(-1) M(-1) for 3. The lack of an N-acetyl group at subsite (-1) reduced the activity to a level 0.1% of that obtained with compound 1, while the absence of the N-acetyl group at subsite (-2) reduced the relative activity to 5.7%. These observations strongly support the theory that chitinase A1 catalysis occurs via a 'substrate-assisted' mechanism. Using these novel fluorogenic substrates, we were able to quantitatively evaluate the recognition specificity of subsite (-2) toward the N-acetyl group of the substrate sugar residue. The (-2) subsite of chitinase A1 was found to specifically recognize an N-acetylated sugar residue, but this specificity was not as strict as that found in subsite (-1).
Asunto(s)
Bacillus/enzimología , Quitinasas/metabolismo , Disacáridos/metabolismo , Acetilación , Disacáridos/química , Himecromona/análogos & derivados , Himecromona/química , Himecromona/metabolismo , Cinética , Especificidad por SustratoRESUMEN
Based on the crystal structure of chitosanase from Streptomyces sp. N174, we have calculated theoretical pK(a) values of the ionizable groups of this protein using a combination of the boundary element method and continuum electrostatics. The pK(a) value obtained for Arg(205), which is located in the catalytic cleft, was abnormally high (>20.0), indicating that the guanidyl group may interact strongly with nearby charges. Chitosanases possessing mutations in this position (R205A, R205H, and R205Y), produced by Streptomyces lividans expression system, were found to have less than 0.3% of the activity of the wild type enzyme and to possess thermal stabilities 4-5 kcal/mol lower than that of the wild type protein. In the crystal structure, the Arg(205) side chain is in close proximity to the Asp(145) side chain (theoretical pK(a), -1.6), which is in turn close to the Arg(190) side chain (theoretical pK(a), 17.7). These theoretical pK(a) values are abnormal, suggesting that both of these residues may participate in the Arg(205) interaction network. Activity and stability experiments using Asp(145)- and Arg(190)-mutated chitosanases (D145A and R190A) provide experimental data supporting the hypothesis derived from the theoretical pK(a) data and prompt the conclusion that Arg(205) forms a strong interaction network with Asp(145) and Arg(190) that stabilizes the catalytic cleft.
Asunto(s)
Arginina/fisiología , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Streptomyces/enzimología , Secuencia de Aminoácidos , Aminoácidos/química , Dominio Catalítico , Dicroismo Circular , Cristalografía por Rayos X , Escherichia coli/metabolismo , Glucosamina/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos/metabolismo , Pliegue de Proteína , Homología de Secuencia de Aminoácido , Temperatura , Termodinámica , Factores de TiempoRESUMEN
After the epoch-making report on X-ray crystal structure of a lysozyme-N-acetylglucosamine trisaccharide complex in 1967, catalytic mechanisms of glycosyl hydrolases have been discussed with reference to the lysozyme mechanism. From the recent findings of chitinolytic enzymes, however, the enzymes were found to have catalytic and substrate binding mechanisms different from those of lysozyme. Based on the X-ray crystal structures of chitinases and their complexes with substrate analogues, the catalytic mechanisms were discussed considering the relative locations of catalytic residues to the bound substrate analogues. Resembling the lysozyme catalytic center, family 19 chitinases, family 46 chitosanases, and family 23 lysozymes have two carboxyl groups at the catalytic center, which are separated (> 10 +) on either side of the catalytic cleft. The catalytic reaction of the enzymes takes place through a single displacement mechanism. In family 18 chitinases, one can identify only one catalytic carboxylate as a proton donor, but not the second catalytic carboxylate whose function and location are similar to those of Asp52 in lysozyme. The catalytic reaction of family 18 chitinases is most likely to take place through a substrate-assisted mechanism. Hen egg white lysozyme has the binding cleft represented by (-4)(-3)(-2)(-1)(+1)(+2). The binding cleft of family 19 chitinases, family 46 chitosanases, and family 23 lysozymes, however, is represented by (-3)(-2)(-1)(+1)(+2)(+3). Molecular dynamics calculation suggests that family 18 chitinases have the binding cleft, (-4)(-3)(-2)(-1)(+1)(+2). The functional diversity of the chitinolytic enzymes might be related to different physiological functions of the enzymes. The enzymes are now being applied to plant protection from fungal pathogens and insect pests. Structure of the targeted chitinous component was determined by a combination of enzyme digestion and solid state CP/MAS NMR spectroscopy, and have been taken into consideration for efficient application of the enzymes. Recent understanding of the catalytic and substrate binding mechanisms would be helpful as well for arrangement of a powerful strategy in such an application.
Asunto(s)
Quitina/metabolismo , Quitinasas/metabolismo , Glicósido Hidrolasas/metabolismo , Muramidasa/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Catálisis , Pollos , Quitinasas/química , Quitinasas/clasificación , Cristalografía por Rayos X , Diseño de Fármacos , Proteínas del Huevo/química , Proteínas del Huevo/metabolismo , Femenino , Gansos , Glicósido Hidrolasas/química , Glicósido Hidrolasas/clasificación , Glicosilación , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Insecticidas/química , Manduca/enzimología , Modelos Moleculares , Muramidasa/química , Muramidasa/clasificación , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
To study the involvement of Asp280 of chitinase A1 from Bacillus circulans WL-12 in the catalytic reaction of the enzyme, site-directed mutagenesis of this residue was carried out. Kinetic analysis of the mutant chitinases revealed that the residue is not absolutely essential for the catalytic reaction and thus, does not act as the catalytic nucleophile.
RESUMEN
4-Methylumbelliferyl beta-chitotrioside [(GlcN)(3)-UMB] was prepared from 4-methylumbelliferyl tri-N-acetyl-beta-chitotrioside [(GlcNAc)(3)-UMB] using chitin deacetylase from Colletotrichum lindemuthianum, and hydrolyzed by chitosanase from Streptomyces sp. N174. The enzymatic deacetylation of (GlcNAc)(3)-UMB was confirmed by (1)H-NMR spectroscopy and mass spectrometry. When the (GlcN)(3)-UMB obtained was incubated with chitosanase, the fluorescence intensity at 450 nm obtained by excitation at 360 nm was found to increase with proportion to the reaction time. The rate of increase in the fluorescence intensity was proportional to the enzyme concentration. This indicates that chitosanase hydrolyzes the glycosidic linkage between a GlcN residue and UMB moiety releasing the fluorescent UMB molecule. Since (GlcN)(3) itself cannot be hydrolyzed by the chitosanase, (GlcN)(3)-UMB is considered to be a useful low molecular weight substrate for the assay of chitosanase. The k(cat) and K(m) values obtained for the substrate (GlcN)(3)-UMB were determined to be 8.1 x 10(-5) s(-1) and 201 microM, respectively. From TLC analysis of the reaction products, the chitosanase was found to hydrolyze not only the linkages between a GlcN residue and UMB moiety, but also the linkages between GlcN residues. Nevertheless, the high sensitivity of the fluorescence detection of the UMB molecule would enable a more accurate determination of kinetic constants for chitosanases.
Asunto(s)
Glicósido Hidrolasas/metabolismo , Trisacáridos/metabolismo , Umbeliferonas/metabolismo , Amidohidrolasas/metabolismo , Secuencia de Carbohidratos , Catálisis , Hidrólisis , Cinética , Datos de Secuencia Molecular , Espectrometría de Fluorescencia , Streptomyces/enzimología , Trisacáridos/química , Umbeliferonas/químicaRESUMEN
Tryptophan residues in chitosanase from Streptomyces sp. N174 (Trp28, Trp101, and Trp227) were mutated to phenylalanine, and thermal unfolding experiments of the proteins were done in order to investigate the role of tryptophan residues in thermal stability. Four types of mutants (W28F, W101F, W227F and W28F/W101F) were produced in sufficient quantity in our expression system using Streptomyces lividans TK24. Each unfolding curve obtained by CD at 222 nm did not exhibit a two-state transition profile, but exhibited a biphasic profile: a first cooperative phase and a second phase that is less cooperative. The single tryptophan mutation decreased the midpoint temperature (Tm) of the first transition phase by about 7 degrees C, and the double mutation by about 11 degrees C. The second transition phase in each mutant chitosanase was more distinct and extended than that in the wild-type. On the other hand, each unfolding curve obtained by tryptophan fluorescence exhibited a typical two-state profile and agreed with the first phase of transition curves obtained by CD. Differential scanning calorimetry profiles of the proteins were consistent with the data obtained by CD. These data suggested that the mutation of individual tryptophan residues would partly collapse the side chain interactions, consequently decreasing Tm and enhancing the formation of a molten globule-like intermediate in the thermal unfolding process. The tryptophan side chains are most likely to play important roles in cooperative stabilization of the protein.
Asunto(s)
Glicósido Hidrolasas/química , Streptomyces/enzimología , Triptófano/química , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Glicósido Hidrolasas/genética , Mutación , Fenilalanina/química , Conformación Proteica , Pliegue de Proteína , Espectrometría de Fluorescencia , Streptomyces/genética , Difracción de Rayos XRESUMEN
Substrate binding subsites of barley chitinase and goose egg white lysozyme were comparatively investigated by kinetic analysis using N-acetylglucosamine oligosaccharide as the substrate. The enzymatic hydrolysis of hexasaccharide was monitored by HPLC, and the reaction time-course was analyzed by the mathematical model, in which six binding subsites (B, C, D, E, F, and G) and bond cleavage between sites D and E are postulated. In this model, all of the possible binding modes of substrate and products are taken into consideration assuming a rapid equilibrium in the oligosaccharide binding processes. To estimate the binding free energy changes of the subsites, time-course calculation was repeated with changing the free energy values of individual subsites, until the calculated time-course was sufficiently fitted to the experimental one. The binding free energy changes of the six binding subsites, B, C, D, E, F and G, which could give a calculated time-course best fitted to the experimental, were 0.0, -5.0, +4.1, -0.5, -3.8, and -2.0 kcal/mol for barley chitinase, and -0.5, -2.2, +4.2, -1.5, -2.6, and -2.8 kcal/mol for goose egg white lysozyme. The binding mode predicted from the p-nitrophenyl-penta-N-acetylchitopentaoside splitting pattern for each enzyme was also analyzed by the identical subsite model. Using the free energy values listed above, the binding mode distribution calculated was fitted to the experimental with a slight modification of free energy value at site G. We concluded that the binding subsite model described above reflects the substantial mechanism of substrate binding for both enzymes. The relatively large disparity in free energy value at site C between these enzymes may be due to the different secondary structures of polypeptide segments interacting with the sugar residue at site C.
Asunto(s)
Quitinasas/metabolismo , Gansos/metabolismo , Hordeum/enzimología , Muramidasa/metabolismo , Semillas/enzimología , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Animales , Sitios de Unión , Cromatografía Líquida de Alta Presión , Clara de Huevo , Hidrólisis , Cinética , Modelos Moleculares , Oligosacáridos/metabolismoRESUMEN
Two types of active chimeric enzymes have been constructed by genetic engineering of chicken cytosolic adenylate kinase (AK) and porcine brain UMP/CMP kinase (UCK): one, designated as UAU, carries an AMP-binding domain of AK in the remaining body of UCK; and the other, designated as AUA, carries a UMP/CMP-binding domain of UCK in the remaining body of AK. Steady-state kinetic analysis of these chimeric enzymes revealed that UAU is 4-fold more active for AMP, 40-fold less active for UMP, and 4-fold less active for CMP than the parental UCK, although AUA has considerably lowered reactivity for both AMP and UMP. Circular dichroism spectra of the two chimeric enzymes suggest that UAU and AUA have similar folding structures to UCK and AK, respectively. Furthermore, proton NMR measurements of the UCK and UAU proteins indicate that significant differences in proton signals are limited to the aromatic region, where an imidazole C2H signal assigned to His31 shows a downfield shift upon conversion of UCK to UAU, and the signals assigned to Tyr49 and Tyr56 in the UMP/CMP-binding domain disappear in UAU. In contrast, AUA has a Tm value about 11 degreesC lower than AK, whereas UAU and UCK have similar Tm values. These results together show that the substrate specificity of nucleoside monophosphate (NMP) kinases can be engineered by the domain exchange, even though the base moiety of NMP appears to be recognized cooperatively by both the NMP-binding domain and the MgATP-binding core domain.
Asunto(s)
Adenilato Quinasa/metabolismo , Nucleósido-Fosfato Quinasa/metabolismo , Adenilato Quinasa/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Unión Competitiva , Pollos , Dicroismo Circular , Estabilidad de Enzimas , Cinética , Datos de Secuencia Molecular , Nucleósido-Fosfato Quinasa/química , Conformación Proteica , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , TemperaturaRESUMEN
Hen lysozyme modified with histamine (HML) and Japanese quail lysozyme (JQL) were treated with immobilized metal ion affinity chromatography to analyze the states of their imidazole groups. When Ni(II) was used as the metal ion immobilized, JQL was strongly retained in a Ni(II)-chelating Sepharose column, while hen lysozyme and HML were hardly retained in the same column. All of these lysozymes have a histidine imidazole group at the 15th position, while JQL has an additional histidine imidazole group at the 103rd position and HML has an additional imidazole group covalently attached to Asp101. Thus, I concluded that the imidazole group at the 103rd position of JQL is exposed to the solvent and recognized by the metal ion, but that the imidazole group attached to Asp101 in HML is localized to a hydrophobic region and not recognized by the metal ion.
Asunto(s)
Histamina/química , Imidazoles/química , Muramidasa/química , Animales , Cationes Bivalentes , Pollos , Cromatografía de Afinidad , Coturnix , Femenino , MetalesRESUMEN
The endochitinase from barley is the archetypal enzyme for a large class of plant-derived antifungal chitinases. The X-ray structure was solved previously in our laboratory and a mechanism of action proposed based on structural considerations. In this manuscript we report the use of a defined soluble substrate, 4-methylumbelliferyl beta-N,N',N"-triacetylchitotrioside, to characterize kinetic parameters of the enzyme. The pH profile shows that activity is controlled by a base with a pKa of 3.9 (Glu 89) and an acid with a pKa of 6.9 (Glu 67). The Km using the synthetic substrate is 33 microM, and the k(cat) is 0.33 min(-1), while the Km for (GlcNAc)4 is 3 microM and k(cat) is 35 min(-1). Binding constants were measured for beta-linked oligomers of N-acetylglucosamine. The monomer does not bind and dissociation constants for the dimer, trimer, and tetramer are 43, 19, and 6 microM, respectively. Analysis of kinetic and dissociation constants proves the mechanism of barley chitinase is consistent with a Bi-Bi kinetic model for hydrolysis, with (GlcNAc)4 and water as substrates and (GlcNAc)2 as products. Substrate cleavage patterns show that (GlcNAc)6 is cleaved in half to (GlcNAc)3 as well as into (GlcNAc)4 and (GlcNAc)2 with almost equal efficiency. NMR analysis of cleavage products confirms that the enzyme proceeds with anomeric inversion of products.
Asunto(s)
Quitinasas/metabolismo , Hordeum/enzimología , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Secuencia de Carbohidratos , Quitina/análogos & derivados , Quitina/metabolismo , Electroforesis Capilar , Colorantes Fluorescentes/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Oligosacáridos/metabolismo , Unión Proteica , Trisacáridos/metabolismoRESUMEN
Oligosaccharide binding to chitosanase from Streptomyces sp. N174 was indirectly evaluated from thermal unfolding experiments of the protein. Thermal unfolding curves were obtained by fluorescence spectroscopy in the presence of D-glucosamine oligosaccharides ((GlcN)n, n = 3, 4, 5, and 6) using the inactive mutant chitosanase in which the catalytic residue, Glu22, is mutated to glutamine (E22Q), aspartic acid (E22D), or alanine (E22A). The midpoint temperature of the unfolding transition (Tm) of E22Q was found to be 44.4 degrees C at pH 7.0. However, the Tm increased upon the addition of (GlcN), by 1.3 degrees C (n = 3), 2.5 degrees C (n = 4), 5.2 degrees C (n = 5), or 7.6 degrees C (n = 6). No appreciable change in Tm was observed when (GlcNAc)6 was added to E22Q. The effect of (GlcN)n on the thermal stability was examined using the other protein, RNase T1, but the oligosaccharide did not affect Tm of the protein. Thus, we concluded that the stabilization effect of (GlcN)n on the chitosanase results from specific binding of the oligosaccharides to the substrate binding cleft. When E22D or E22A was used instead of E22Q, the increases in Tm induced by (GlcN)6 binding were 2.7 degrees C for E22D and 4.2 degrees C for E22A. In E22D or E22A, interaction with (GlcN)6 seems to be partly disrupted by a conformational distortion in the catalytic cleft.
Asunto(s)
Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Oligosacáridos/metabolismo , Pliegue de Proteína , Streptomyces/enzimología , Acetilglucosamina/análogos & derivados , Sitios de Unión , Glucosamina/análogos & derivados , Glicósido Hidrolasas/genética , Mutación/genética , Unión Proteica , Espectrometría de Fluorescencia , Streptomyces/genética , Temperatura , Termodinámica , Triptófano/metabolismoRESUMEN
Novel information on the structure and function of chitosanase, which hydrolyzes the beta-1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation.
Asunto(s)
Glicósido Hidrolasas/química , Glicósido Hidrolasas/fisiología , Streptomyces/enzimología , Secuencia de Aminoácidos , Quitinasas/química , Quitinasas/metabolismo , Quitinasas/fisiología , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Muramidasa/química , Muramidasa/metabolismo , Muramidasa/fisiología , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
The cell of Fusarium oxysporum was digested with commercial Bacillus chitosanase. The chitosanase produced low molecular weight heterooligosaccharides consisting of GlcN and GlcNAc from the cell wall. A main component of the digestion products was identified as 2-amino-2-deoxy-beta-D-glucopyranosyl- (1-->4)-2-acetamido-2-deoxy-D-glucopyranose. The chitosanase appeared to be more effective than Streptomyces griseus chitinase for cell wall digestion. Moreover, maltose was unexpectedly found in the digestion products, indicating that the cell wall contains alpha-1,4-linked glucan chain as a polysaccharide component.
Asunto(s)
Acetilglucosamina/análisis , Pared Celular/química , Quitina/análisis , Fusarium/química , Glucosamina/análisis , Oligosacáridos/análisis , Bacillus/enzimología , Fusarium/ultraestructura , Glicósido Hidrolasas , Maltosa/análisis , Estructura MolecularRESUMEN
The comparison of four sequences of prokaryotic chitosanases, belonging to the family 46 of glycosyl hydrolases, revealed a conserved N-terminal module of 50 residues, including five invariant carboxylic residues. To verify if some of these residues are important for catalytic activity in the chitosanase from Streptomyces sp. N174, these 5 residues were replaced by site-directed mutagenesis. Substitutions of Glu-22 or Asp-40 with sterically conservative (E22Q, D40N) or functionally conservative (E22D, D40E) residues reduced drastically specific activity and kcat, while Km was only slightly changed. The other residues examined, Asp-6, Glu-36, and Asp-37, retained significant activity after mutation. Circular dichroism studies of the mutant chitosanases confirmed that the observed effects are not due to changes in secondary structure. These results suggested that Glu-22 and Asp-40 are directly involved in the catalytic center of the chitosanase and the other residues are not essential for catalytic activity.
Asunto(s)
Evolución Biológica , Glicósido Hidrolasas/metabolismo , Streptomyces/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Ácidos Carboxílicos/metabolismo , Catálisis , Dicroismo Circular , Clonación Molecular , Secuencia Conservada , Escherichia coli/genética , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/aislamiento & purificación , Hidrólisis , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligosacáridos/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Chitosanase was produced by the strain of Streptomyces lividans TK24 bearing the csn gene from Streptomyces sp. N174, and purified by S-Sepharose and Bio-Gel A column chromatography. Partially (25-35%) N-acetylated chitosan was digested by the purified chitosanase, and structures of the products were analysed by NMR spectroscopy. The chitosanase produced heterooligosaccharides consisting of D-GlcN and GlcNAc in addition to glucosamine oligosaccharides [(GlcN)n, n = 1, 2 and 3]. The reducing- and non-reducing-end residues of the heterooligosaccharide products were GlcNAc and GlcN respectively, indicating that the chitosanase can split the GlcNAc-GlcN linkage in addition to that of GlcN-GlcN. Time-dependent 1H-NMR spectra showing hydrolysis of (GlcN)6 by the chitosanase were obtained in order to determine the anomeric form of the reaction products. The chitosanase was found to produce only the alpha-form; therefore it is an inverting enzyme. Separation and quantification of (GlcN)n was achieved by HPLC, and the time course of the reaction catalysed by the chitosanase was studied using (GlcN)n (n = 4, 5 and 6) as the substrate. The chitosanase hydrolysed (GlcN)6 in an endo-splitting manner producing (GlcN)2, (GlcN)3 and (GlcN)4, and did not catalyse transglycosylation. Product distribution was (GlcN)3 >> (GlcN)2 > (GlcN)4. Cleavage to (GlcN)3 + (GlcN)3 predominated over that to (GlcN)2 + (GlcN)4. Time courses showed a decrease in rate of substrate degradation from (GlcN)6 to (GlcN)5 to (GlcN)4. It is most likely that the substrate-binding cleft of the chitosanase can accommodate at least six GlcN residues, and that the cleavage point is located at the midpoint of the binding cleft.