Muramidase Catalyzed Hydrolysis of Glycol Chitin

The mode of action of muramidase on glycol chitin was investigated following the degradation of glycol chitin by the estimation of reducing power produced by hydrolysis. The optimum temperature as well as pH for the muramidase activity were found to lie at 50°C and pH 5 respectively. These values nearly agree with those obtained by α viscosimetric determination of muramidase activity.

There are, however, some difficulties in the applicability of reducing power estimating method as an assay of muramidase, since the hydrolysis of glycol chitin catalyzed by muramidase seems to be rather of complicated; for instance, the pH values, temperature and E/S ratio remarkably affect the final value of reducing power produced from a known amount of substrate.

Therefore, experiments were carried out to elucidate such anomalous phenomena and the mode of hydrolysis is discussed.     

Mutation of Trp137 to glutamate completely removes transglycosyl activity associated with the <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> AfChiB1

Family 18 chitinases hydrolyze chitin through a substrate-assisted catalytic mechanism and are to a variable extent able to catalyze transglycosylation reactions. Previously Aspergillus fumigatus AfChiB1 was found to be able to catalyze transglycosylation reactions. Structural analysis reveals that AfChiB1 consists of an eight-stranded β/α-barrel. Like other members of the family 18 hydrolases, AfChiB1 has conserved substrate binding site and catalytic acid, while a suitable nucleophile is missing. In this study, Trp137, Asp246, and Met243, which are close to the glycosidic cleavage site, were mutated to glutamate individually. As a result, the W137E remained its hydrolytic activity and was completely devoid of transglycosyl activity, while the D246E reduced its chitinolytic activity and increased its transglycosyl activity. And the M243E showed a remarkable reduction of chitinolytic activity and complete loss of transglycosyl activity. These results suggested that the transglycosyl reaction catalyzed by the AfChiB1 is due to lacking of nucleophile. Enzymes: exochitinases (EC 3.2.1.14)     

Hydrolysis of glycol chitin by chitinolytic enzymes

• 1.1. The hydrolysis of glycol chitin preparations by several β-N-acetylglucosaminidases was monitored colorimetrically with the potassium ferriferrocyanide reagent.
• 2.2. Glycol chitin samples from crab and insect sources varied considerably in chemical composition and susceptibility to enzymatic hydrolysis.
• 3.3. Insect endochitinase preferred crab glycol chitin as substrate while hen's egg white lysozyme preferred commercial glycol chitin.
• 4.4. Insect glycol chitin was well hydrolyzed by both enzymes.
• 5.5. Insect exochitinase did not digest glycol chitin.

     

Studies on the Chitinolytie Enzymes of Black-koji Mold

In an attempt to separate the enzyme system participating in the decomposition of glycol chitin to constituent aminosugar, the purification of chitinase of Aspergillus niger was carried out by detemining both liquefying and saccharifying activities. Using fractionation with ammonium sulfate and column chromatography by hydroxylapatite, the chitinase system of the mold was separated into different enzyme fractions, which were required for the complete hydrolysis of glycol chitin. It was found that one of these enzymes caused a rapid decrease in viscosity of glycol chitin solution, another enzyme possessed N-acetyl-β-glucosaminidase activity upon N, N′-diacetylchitobiose and β-methyl-N-acetylglucosaminide, and that glycol chitin was decomposed to constituent aminosugar by a successive action of the two different enzymes.     

Difference in substrate binding processes between intact and iodine-inactivated lysozyme.

The binding of glycol chitin to intact and iodine-inactivated lysozyme was studied by measuring the absorbance of the complex with N-methylnicotinamide chloride, which binds to the subsite C in lysozyme as a competitive inhibitor. The association constant of glycol chitin to inactivated lysozyme was determined from static experiments to be 1.7 × 10⁴M^?1. The kinetics of the substrate binding to intact and iodine-inactivated lysozyme were measured by the stopped-flow method at 23°C and pH 5.6. The binding to inactivated lysozyme was clearly monophasic, whereas in intact lysozyme it consisted of multiple phases. In the substrate binding to intact lysozyme, a fast bimolecular process and two subsequent slow unimolecular processes were observed besides the hydrolysis process of polymer substrate. These slow phases were missing completely in inactivated lysozyme. It results from the alteration in the local structure occurring at the subsite D in inactivated lysozyme. These results mean that the slow phases are important for catalytic action of lysozyme. The rate constants of association and dissociation in the fast bimolecular process were determined in this paper. Furthermore, the association constant of the substrate to intact lysozyme was also determined kinetically to be 6.5 × 10³M^?1.     

Mechanism of initial rapid rate retardation in cellobiohydrolase catalyzed cellulose hydrolysis

Despite intensive research, the mechanism of the rapid retardation in the rates of cellobiohydrolase (CBH) catalyzed cellulose hydrolysis is still not clear. Interpretation of the hydrolysis data has been complicated by the inability to measure the catalytic constants for CBH‐s acting on cellulose. We developed a method for measuring the observed catalytic constant (k^obs) for CBH catalyzed cellulose hydrolysis. It relies on in situ measurement of the concentration of CBH with the active site occupied by the cellulose chain. For that we followed the specific inhibition of the hydrolysis of para‐nitrophenyl‐β‐D ‐lactoside by cellulose. The method was applied to CBH‐s TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium and their isolated catalytic domains. Bacterial microcrystalline cellulose, Avicel, amorphous cellulose, and lignocellulose were used as substrates. A rapid decrease of k^obs in time was observed on all substrates. The k^obs values for PcCel7D were about 1.5 times higher than those for TrCel7A. In case of both TrCel7A and PcCel7D, the k^obs values for catalytic domains were similar to those for intact enzymes. A model where CBH action is limited by the average length of obstacle‐free way on cellulose chain is proposed. Once formed, productive CBH–cellulose complex proceeds with a constant rate determined by the true catalytic constant. After encountering an obstacle CBH will “get stuck” and the rate of further cellulose hydrolysis will be governed by the dissociation rate constant (k_off), which is low for processive CBH‐s. Biotechnol. Bioeng. 2010;106: 871–883. © 2010 Wiley Periodicals, Inc.     

Interchange of functional domains switches enzyme specificity: construction of a chimeric pneumococcal-clostridial cell wall lytic enzyme

Bacterial autolysins are endogenous enzymes that specifically cleave covalent bonds in the cell wall. These enzymes show both substrate and bond specificities. The former is related to their interaction with the insoluble substrate whereas the latter determine their site of action. The bond specificity allows their classification as muramidases (lysozymes), glucosaminldases, amidases, and endopeptidases. To demonstrate that the autolysin (LYC muramidase) of Clostridium acetobutylicum ATCC824 presents a domainal organization, a chimeric gene (clc) containing the regions coding for the catalytic domain of the LYC muramidase and the choline-binding domain of the pneumococcal phage CPL1 muramidase has been constructed by in vitro recombination of the corresponding gene fragments. This chimeric construction codes for a choline-binding protein (CLC) that has been purified using affinity chromatography on DEAE-cellulose. Several biochemical tests demonstrate that this rearrangement of domains has generated an enzyme with a choline-dependent muramidase activity on pneumococcal cell walls. Since the parental LYC muramidase was cholineindependent and unable to degrade pneumococcal cell walls, the formation of this active chimeric enzyme by exchanging protein domains between two enzymes that specifically hydrolyse cell walls of bacteria belonging to different genera shows that a switch on substrate specificity has been achieved. The chimeric CLC muramidase behaved as an autolytic enzyme when it was adsorbed onto a live autolysin-defective mutant of Streptococcus pneumoniae. The construction described here provides experimental support for the theory of modular evolution which assumes that novel proteins have evolved by the assembly of preexisting polypeptide units.     

Importance of Trp59 and Trp60 in chitin-binding, hydrolytic, and antifungal activities of Streptomyces griseus chitinase C

The chitin-binding domain of Streptomyces griseus chitinase C (ChBD_ChiC) belongs to CBM family 5. Only two exposed aromatic residues, W59 and W60, were observed in ChBD_ChiC, in contrast to three such residues on CBD_Cel5 in the same CBM family. To study importance of these residues in binding activity and other functions of ChBD_ChiC, site-directed mutagenesis was carried out. Single (W59A and W60A) and double (W59A/W60A) mutations abolished the binding activity of ChiC to colloidal chitin and decreased the hydrolytic activity toward not only colloidal chitin but also a soluble high Mr substrate, glycol chitin. Interaction of ChBD_ChiC with oligosaccharide was eliminated by these mutations. The hydrolytic activity toward oligosaccharide was increased by deletion of ChBD but not affected by these mutations, indicating that ChBD interferes with oligosaccharide hydrolysis but not through its binding activity. The antifungal activity was drastically decreased by all mutations and significant difference was observed between single and double mutants. Taken together with the structural information, these results suggest that ChBD_ChiC binds to chitin via a mechanism significantly different from CBD_Cel5, where two aromatic residues play major role, and contributes to various functions of ChiC. Sequence comparison indicated that ChBD_ChiC-type CBMs are dominant in CBM family 5.     

Purification and Characterization of Extracellular Chitin Deacetylase from Colletotrichum lindemuthianum

Chitin deacetylase, active in the presence of acetate (96% of the enzymatic activity was retained in the presence of 100 mm sodium acetate), was purified to electrophoretic homogeneity from a culture filtrate of Colletotrichum lindemuthianum (944-fold with a recovery of 4.05%). The enzyme was induced in the medium after the eighth day of incubation simultaneously with the blackening of the medium. The molecular mass of the enzyme was 31.5 kDa and 33 kDa as judged by SDS–PAGE and gel filtration, respectively, suggesting that the enzyme is a single polypeptide. The optimum temperature was 60°C and the optimum pH was 11.5–12.0 when glycol chitin was used as substrate. The enzyme was active toward glycol chitin, partially N-deacetylated water soluble chitin, and chitin oligomers the degrees of polymerization of which were more than four, but was less active with chitin trimer and dimer, and inactive with N-acetylglucosamine. The K_m and k_cat for glycol chitin were 2.55 mm and 27.1s^?1, respectively, and those for chitin pentamer were 414 μm and 83.2s^?1, respectively. The reaction rates of the enzyme toward glycol chitin and chitin oligomers seemed to follow the Michaelis–Menten kinetics.     

Mechanistic Studies Reveal Similar Catalytic Strategies for Phosphodiester Bond Hydrolysis by Protein-only and RNA-dependent Ribonuclease P

Ribonuclease P (RNase P) is an endonuclease that catalyzes the essential removal of the 5′ end of tRNA precursors. Until recently, all identified RNase P enzymes were a ribonucleoprotein with a conserved catalytic RNA component. However, the discovery of protein-only RNase P (PRORP) shifted this paradigm, affording a unique opportunity to compare mechanistic strategies used by naturally evolved protein and RNA-based enzymes that catalyze the same reaction. Here we investigate the enzymatic mechanism of pre-tRNA hydrolysis catalyzed by the NYN (Nedd4-BP1, YacP nuclease) metallonuclease of Arabidopsis thaliana, PRORP1. Multiple and single turnover kinetic data support a mechanism where a step at or before chemistry is rate-limiting and provide a kinetic framework to interpret the results of metal alteration, mutations, and pH dependence. Catalytic activity has a cooperative dependence on the magnesium concentration (n_H = 2) under k_cat/K_m conditions, suggesting that PRORP1 catalysis is optimal with at least two active site metal ions, consistent with the crystal structure. Metal rescue of Asp-to-Ala mutations identified two aspartates important for enhancing metal ion affinity. The single turnover pH dependence of pre-tRNA cleavage revealed a single ionization (pK_a ∼ 8.7) important for catalysis, consistent with deprotonation of a metal-bound water nucleophile. The pH and metal dependence mirrors that observed for the RNA-based RNase P, suggesting similar catalytic mechanisms. Thus, despite different macromolecular composition, the RNA and protein-based RNase P act as dynamic scaffolds for the binding and positioning of magnesium ions to catalyze phosphodiester bond hydrolysis.     

Purification and Characterization of Chitosanase and Exo-β-D-Glucosaminidase from a Koji Mold,Aspergillus oryzae IAM2660

Chitosan-degrading activity was detected in the culture fluid of Aspergillus oryzae, A. sojae, and A. flavus among various fungal strains belonging to the genus Aspergillus. One of the strong producers, A. oryzae IAM2660 had a higher level of chitosanolytic activity when N-acetylglucosamine (GlcNAc) was used as a carbon source. Two chitosanolytic enzymes, 40 kDa and 135 kDa in molecular masses, were purified from the culture fluid of A. oryzae IAM2660. Viscosimetric assay and an analysis of reaction products by thin-layer chromatography clearly indicated the endo- and exo-type cleavage manner for the 40-kDa and 135-kDa enzymes, respectively. The 40-kDa enzyme, designated chitosanase, catalyzed a hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, glycol chitosan, and chitosan with a low degree of acetylation (0-30%). The 135-kDa enzyme, named exo-β-D-glucosaminidase, released a single GlcN residue from the GlcN oligomers and chitosan, but did not release GlcNAc residues from either GlcNAc oligomer or colloidal chitin.     

On the mechanism of citrate inhibition of ceruloplasmin ferroxidase activity

Ceruloplasmin is a plasma protein, which oxidizes ferrous ions in a catalytic manner. It is considered to function as a ferroxidasein vivo. Citrate was found to inhibit the reaction. The ceruloplasmin catalyzed oxidation ofp-phenylenediamines, however, was not affected by citrate. The inhibitory effect is proposed to be due to formation of Fe²⁺-citrate, which does not react with ceruloplasmin. The stability constant for the Fe²⁺-citrate complex estimated from the present inhibition study is in good agreement with previously published data.     

Critical Active-Site Residues Identified by Site-Directed Mutagenesis in Pseudomonas aeruginosa Phosphorylcholine Phosphatase, A New Member of the Haloacid Dehalogenases Hydrolase Superfamily

Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP), the product of the PA5292 gene, is synthesized when the bacteria are grown with choline, betaine, dimethylglycine, or carnitine. In the presence of Mg²⁺, PChP catalyzes the hydrolysis of both phosphorylcholine (PCh) and p-nitrophenylphosphate (p-NPP). PCh saturation curve analysis of the enzyme with or without the signal peptide indicated that the peptide was the fundamental factor responsible for decreasing the affinity of the second site of PChP for PCh, either at pH 5.0 or pH 7.4. PChP contained three conserved motifs characteristic of the haloacid dehalogenases superfamily. In the PChP without the signal peptide, motifs I, II, and III correspond to the residues ³¹DMDNT³⁵, ¹⁶⁶SAA¹⁶⁸, and K²⁴²/²⁶¹GDTPDSD²⁶⁷, respectively. To determine the catalytic importance of the D31, D33, T35, S166, K242, D262, D265, and D267 on the enzyme activity, site-directed mutagenesis was performed. D31, D33, D262, and D267 were identified as the more important residues for catalysis. D265 and D267 may be involved in the stabilization of motif III, or might contribute to substrate specificity. The substitution of T35 by S35 resulted in an enzyme with a low PChP activity, but conserves the catalytic sites involved in the hydrolysis of PCh (K_m1 0.03 mM, K_m2 0.5 mM) or p-NPP (K_m 2.1 mM). Mutating either S166 or K242 revealed that these residues are also important to catalyze the hydrolysis of both substrates. The substitution of lysine by arginine or by glutamine revealed the importance of the positive charged group, either from the amino or guanidinium groups, because K242Q was inactive, whereas K242R was a functional enzyme.     

昆虫几丁质合成酶及其抑制剂

几丁质合成酶(CS)是几丁质合成的关键酶,它具有3个结构域:结构域A、结构域B和结构域C,其中结构域B是催化域。根据氨基酸序列的差异,几丁质合成酶分为两类:CS-A及CS-B,分别在表皮及围食膜基质中催化合成几丁质。关于几丁质合成有2种假想模型。有多种抑制剂可以抑制几丁质的合成,其中核苷肽抗生素类及核苷磷酸类作用于CS的催化部位,是竞争性抑制剂,其它抑制剂的作用机理仍不明确。     

An extracellular Bacillus sp. chitinase for the production of chitotriose as a major chitinolytic product

An extracellular chitinase of Bacillus sp. WY22 was purified by 9.6-fold. It had a Mr of 35 kDa, an apparent K _m value for colloidal chitin of 3 mg ml^–1 and was optimally active at 37 °C and pH 5.5 over 1 h. The enzyme could also hydrolyse swollen chitin, glycol chitin and chitosan with relative activities of 76%, 34% and 23% compared with colloidal chitin. It formed chitotriose as a major product from colloidal chitin and glycol chitin.     

Insect chitin synthases: a review

Chitin is the most widespread amino polysaccharide in nature. The annual global amount of chitin is believed to be only one order of magnitude less than that of cellulose. It is a linear polymer composed of N-acetylglucosamines that are joined in a reaction catalyzed by the membrane-integral enzyme chitin synthase, a member of the family 2 of glycosyltransferases. The polymerization requires UDP–N-acetylglucosamines as a substrate and divalent cations as co-factors. Chitin formation can be divided into three distinct steps. In the first step, the enzymes‘ catalytic domain facing the cytoplasmic site forms the polymer. The second step involves the translocation of the nascent polymer across the membrane and its release into the extracellular space. The third step completes the process as single polymers spontaneously assemble to form crystalline microfibrils. In subsequent reactions the microfibrils combine with other sugars, proteins, glycoproteins and proteoglycans to form fungal septa and cell walls as well as arthropod cuticles and peritrophic matrices, notably in crustaceans and insects. In spite of the good effort by a hardy few, our present knowledge of the structure, topology and catalytic mechanism of chitin synthases is rather limited. Gaps remain in understanding chitin synthase biosynthesis, enzyme trafficking, regulation of enzyme activity, translocation of chitin chains across cell membranes, fibrillogenesis and the interaction of microfibrils with other components of the extracellular matrix. However, cumulating genomic data on chitin synthase genes and new experimental approaches allow increasingly clearer views of chitin synthase function and its regulation, and consequently chitin biosynthesis. In the present review, I will summarize recent advances in elucidating the structure, regulation and function of insect chitin synthases as they relate to what is known about fungal chitin synthases and other glycosyltransferases.     

Enantioselective hydrolysis of β-hydroxy nitriles using the whole cell biocatalyst Rhodococcus rhodochrous ATCC BAA-870

Candida rugosa lipase was covalently immobilized onto silica gel in two different ways: via glutaraldehyde (L_GAL) and via hydrophobic spacer arm (1,6 diamino hexane) (L_SA). Free lipase, L_GAL and L_SA were used to investigate the hydrolysis of two different substrates, namely p-nitrophenyl palmytate (pNPP) and p-nitrophenyl acetate (pNPA), both in aqueous medium. In addition, these lipase samples were used to synthesize the pNPP from p-nitrophenol (pNP) and palmytic acid (PA) and pNPA from pNP and acetic acid (AA), both in hexane medium. Hydrolytic and synthetic activities of L_SA were higher than those of free lipase and L_GAL. Synthetic activities of free lipase, L_GAL and L_SA for pNPA in the presence of pNP and AA within hexane medium were higher than those of hydrolytic activities for pNPA in aqueous medium. The same tendency was also observed with pNPP. The effects of pH and temperature on hydrolytic and synthetic activities were investigated for all lipase preparations. Operational stability was the highest for L_GAL and L_SA when these enzymes were used for pNPP synthesis and in hexane medium, after 100 repeated uses, 68% and 51% of initial activities remained, respectively, at the end of 100 repeated cycles. Free lipase lost all of its activity within 15 and 20 days when stored at 25 °C and 5 °C, respectively. However, L_GAL showed 54% and 70% of initial activity at the end of 60 storage days at 25 °C and 5 °C, respectively, while these values were observed as 36% and 60% for L_SA.     

Contribution of water to free energy of hydrolysis of pyrophosphate

The energy of hydrolysis of phosphate compounds varies depending on whether they are in solution or bound to the catalytic site of enzymes. With the purpose of simulating the conditions at the catalytic site, the observed equilibrium constant for pyrophosphate hydrolysis (Kobsd) was measured in aqueous mixtures of dimethyl sulfoxide, ethylene glycol, or polymers of ethylene glycol. The reaction was catalyzed by yeast inorganic pyrophosphatase at 30 degrees C. All the cosolvents used promoted a decrease of Kobsd. Polymers of ethylene glycol were more effective than dimethyl sulfoxide or ethylene glycol in decreasing Kobsd. The higher the molecular weight of the polymer, the lower the value of Kobsd. A decrease in Kobsd from 346 M (delta G degree obsd = -3.5 kcal mol-1) to 0.1 M (delta G degree obsd = 1.3 kcal mol-1) was observed after the addition of 50% (w/v) poly(ethylene glycol) 8000 to a solution containing 0.9 mM MgCl2 and 1 mM Pi at pH 8.0. The association constants of Pi and pyrophosphate for H+ and Mg2+ were measured in presence of different ethylene glycol concentrations in order to calculate the Keq for hydrolysis of different ionic species of pyrophosphate. A decrease in all the Keq was observed. The results are interpreted according to the concept that the energy of hydrolysis of phosphate compounds depends on the different solvation energies of reactants and products.     

Preparation and characterization of an active lysozyme derivative: Kyn 62-lysozyme.

A novel method for the preparation of Kyn 62-lysozyme, in which tryptophan 62 is replaced by kynurenine, is reported. Hen egg-white lysozyme was ozonized in aqueous solution to yield one N'-formylkynurenine residue and deformylated with hydrochloric acid in frozen solution at -10 degrees C. Crude Kyn 62-lysozyme was purified by affinity and Bio Rex 70 chromatography successively. Kyn 62-lysozyme retains affinity for chitin and is essentially an active enzyme with a slightly weakened but distinct catalytic activity. After this modification, the enzyme activity was changed differently depending on the kind of substrate. At the individual optimum pH's, lytic activity was largely retained (80% active), but the catalytic efficiency for hydrolyzing glycol chitin was relatively low (30% active). Lysis of M. lysodeikticus cell suspensions was optimally catalyzed by Kyn 62-lysozyme at pH 6.2 and at 0.088 ionic strength. These values are lower by 1.3 pH unit and 0.04 ionic strength, respectively, than those of intact lysozyme. The optimum pH and ionic strength for the hydrolysis of neutral substrates were scarcely affected. These results suggest the significance of electrostatic interaction in the lysis of lysozyme. Relatively limited loss of activity induced by modification of the 62nd residue, which is thought to participate directly in the binding of the substrate at subsite C, is discussed on the basis of the similarity of side chain structure in tryptophan and kynurenine.     

Characterization of the First Fungal Glycosyl Hydrolase Family 19 Chitinase (NbchiA) from Nosema bombycis (Nb)

Chitinases (EC 3.2.1.14), as one kind of glycosyl hydrolase, hydrolyze the β‐(1,4) linkages of chitin. According to the sequence similarity, chitinases can be divided into glycoside hydrolase family 18 and family 19. Here, a chitinase from Nosema bombycis (NbchiA) was cloned and purified by metal affinity chromatography and molecular exclusion chromatography. Sequence analysis indicated that NbchiA belongs to glycoside hydrolase family 19 class IV chitinase. The optimal pH and temperature of NbchiA are 7.0 and 40 °C, respectively. This purified chitinase showed high activity toward soluble substrates such as ethylene glycol chitin and soluble chitosan. The degradation of chitin oligosaccharides (GlcNAc)_2–5 detected by high‐performance liquid chromatography showed that NbchiA hydrolyzed mainly the second glycosidic linkage from the reducing end of (GlcNAc)_3‐5. On the basis of structure‐based multiple‐sequence alignment, Glu51 and Glu60 are believed to be the key catalytic residues. The site‐directed mutation analysis revealed that the enzymatic activity was decreased upon mutation of Glu60, whereas mutation of Glu51 totally abolished the enzymatic activity. This is the first report of a GH19 chitinase in fungi and in Microsporidia.