Kinetic studies on the hydrolysis of N-acetylated and N-deacetylated derivatives of 4-methylumbelliferyl chitobioside by the family 18 chitinases ChiA and ChiB from Serratia marcescens

Kinetic analyses of the hydrolysis reactions of N-acetylated and N-deacetylated derivatives of 4-methylumbelliferyl chitobioside [(GlcNAc)(2)-UMB (1), GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)(2)-UMB (4)] by ChiA and ChiB from Serratia marcescens were performed. Both enzymes released UMB from all compounds apart from 4. The S-v curves of the hydrolyses of 1 by ChiA and ChiB both exhibited atypical kinetic patterns, and the shapes of the two S-v curves were different from one another. However, both curve shapes were explained by assuming some of the enzyme present formed complexes with multiple molecules of the substrate. Conversely, the S-v curves generated in the cleavage of 2 and 3 by ChiA exhibited typical Michaelis-Menten profiles. Both enzymes hydrolysed 2 with an approximately 14-fold higher K(m) value relative to 1, indicating that the N-acetyl group was recognised at the -2 subsite. The k(cat) value obtained with ChiA was identical to the k(cat) value observed for 1. However, the k(cat) value for ChiB was one-fourth that of 1, suggesting that the removal of the N-acetyl group caused an increase in the formation of a non-productive ES-complex. ChiA and ChiB hydrolysed 3 with 5- and 20-fold greater K(m) values relative to 1, respectively, and 60- and 30-fold smaller k(cat) values relative to 1, respectively. The reaction mechanism of family 18 chitinases is discussed based upon the results obtained from the hydrolysis of these compounds.     

X-ray crystallographic study of xylopentaose binding to Pseudomonas fluorescens xylanase A

The structure of the complex between a catalytically compromised family 10 xylanase and a xylopentaose substrate has been determined by X-ray crystallography and refined to 3.2 A resolution. The substrate binds at the C-terminal end of the eightfold betaalpha-barrel of Pseudomonas fluorescens subsp. cellulosa xylanase A and occupies substrate binding subsites -1 to +4. Crystal contacts are shown to prevent the expected mode of binding from subsite -2 to +3, because of steric hindrance to subsite -2. The loss of accessible surface at individual subsites on binding of xylopentaose parallels well previously reported experimental measurements of individual subsites binding energies, decreasing going from subsite +2 to +4. Nine conserved residues contribute to subsite -1, including three tryptophan residues forming an aromatic cage around the xylosyl residue at this subsite. One of these, Trp 313, is the single residue contributing most lost accessible surface to subsite -1, and goes from a highly mobile to a well-defined conformation on binding of the substrate. A comparison of xylanase A with C. fimi CEX around the +1 subsite suggests that a flatter and less polar surface is responsible for the better catalytic properties of CEX on aryl substrates. The view of catalysis that emerges from combining this with previously published work is the following: (1) xylan is recognized and bound by the xylanase as a left-handed threefold helix; (2) the xylosyl residue at subsite -1 is distorted and pulled down toward the catalytic residues, and the glycosidic bond is strained and broken to form the enzyme-substrate covalent intermediate; (3) the intermediate is attacked by an activated water molecule, following the classic retaining glycosyl hydrolase mechanism.     

High resolution structural analyses of mutant chitinase A complexes with substrates provide new insight into the mechanism of catalysis

Chitinase A (ChiA) from the bacterium Serratia marcescens is a hydrolytic enzyme, which cleaves beta-1,4-glycosidic bonds of the natural biopolymer chitin to generate di-N-acetyl-chitobiose. The refined structure of ChiA at 1.55 A shows that residue Asp313, which is located near the catalytic proton donor residue Glu315, is found in two alternative conformations of equal occupancy. In addition, the structures of the cocrystallized mutant proteins D313A, E315Q, Y390F, and D391A with octa- or hexa-N-acetyl-glucosamine have been refined at high resolution and the interactions with the substrate have been characterized. The obtained results clearly show that the active site is a semiclosed tunnel. Upon binding, the enzyme bends and rotates the substrate in the vicinity of the scissile bond. Furthermore, the enzyme imposes a critical "chair" to "boat" conformational change on the sugar residue bound to the -1 subsite. According to our results, we suggest that residues Asp313 and Tyr390 along with Glu315 play a central role in the catalysis. We propose that after the protonation of the substrate glycosidic bond, Asp313 that interacts with Asp311 flips to its alternative position where it interacts with Glu315 thus forcing the substrate acetamido group of -1 sugar to rotate around the C2-N2 bond. As a result of these structural changes, the water molecule that is hydrogen-bonded to Tyr390 and the NH of the acetamido group is displaced to a position that allows the completion of hydrolysis. The presented results suggest a mechanism for ChiA that modifies the earlier proposed "substrate assisted" catalysis.     

Subsite mapping of Aspergillus niger endopolygalacturonase II by site-directed mutagenesis

To assess the subsites involved in substrate binding in Aspergillus niger endopolygalacturonase II, residues located in the potential substrate binding cleft stretching along the enzyme from the N to the C terminus were subjected to site-directed mutagenesis. Mutant enzymes were characterized with respect to their kinetic parameters using polygalacturonate as a substrate and with respect to their mode of action using oligogalacturonates of defined length (n = 3-6). In addition, the effect of the mutations on the hydrolysis of pectins with various degrees of esterification was studied. Based on the results obtained with enzymes N186E and D282K it was established that the substrate binds with the nonreducing end toward the N terminus of the enzyme. Asn(186) is located at subsite -4, and Asp(282) is located at subsite +2. The mutations D183N and M150Q, both located at subsite -2, affected catalysis, probably mediated via the sugar residue bound at subsite -1. Tyr(291), located at subsite +1 and strictly conserved among endopolygalacturonases appeared indispensable for effective catalysis. The mutations E252A and Q288E, both located at subsite +2, showed only slight effects on catalysis and mode of action. Tyr(326) is probably located at the imaginary subsite +3. The mutation Y326L affected the stability of the enzyme. For mutant E252A, an increased affinity for partially methylesterified substrates was recorded. Enzyme N186E displayed the opposite behavior; the specificity for completely demethylesterified regions of substrate, already high for the native enzyme, was increased. The origin of the effects of the mutations is discussed.     

Opposing influences by subsite -1 and subsite +1 residues on relative xylopyranosidase/arabinofuranosidase activities of bifunctional β-D-xylosidase/α-L-arabinofuranosidase

Conformational inversion occurs 7-8kcal/mol more readily in furanoses than pyranoses. This difference is exploited here to probe for active-site residues involved in distorting pyranosyl substrate toward reactivity. Spontaneous glycoside hydrolysis rates are ordered 4-nitrophenyl-α-l-arabinofuranoside (4NPA)>4-nitrophenyl-β-d-xylopyranoside (4NPX)>xylobiose (X2). The bifunctional β-d-xylosidase/α-l-arabinofuranosidase exhibits the opposite order of reactivity, illustrating that the enzyme is well equipped in using pyranosyl groups of natural substrate X2 in facilitating glycoside hydrolysis. Probing the roles of all 17 active-site residues by single-site mutation to alanine and by changing both moieties of substrate demonstrates that the mutations of subsite -1 residues decrease the ratio k(cat)(4NPX/4NPA), suggesting that the native residues support pyranosyl substrate distortion, whereas the mutations of subsite +1 and the subsite -1/+1 interface residues increase the ratio k(cat)(4NPX/4NPA), suggesting that the native residues support other factors, such as C1 migration and protonation of the leaving group. Alanine mutations of subsite -1 residues raise k(cat)(X2/4NPX) and alanine mutations of subsite +1 and interface residues lower k(cat)(X2/4NPX). We propose that pyranosyl substrate distortion is supported entirely by native residues of subsite -1. Other factors leading to the transition state are supported entirely by native residues of subsite +1 and interface residues.     

Tyrosine 105 and threonine 212 at outermost substrate binding subsites -6 and +4 control substrate specificity, oligosaccharide cleavage patterns, and multiple binding modes of barley alpha-amylase 1

The role in activity of outer regions in the substrate binding cleft in alpha-amylases is illustrated by mutational analysis of Tyr(105) and Thr(212) localized at subsites -6 and +4 (substrate cleavage occurs between subsites -1 and +1) in barley alpha-amylase 1 (AMY1). Tyr(105) is conserved in plant alpha-amylases whereas Thr(212) varies in these and related enzymes. Compared with wild-type AMY1, the subsite -6 mutant Y105A has 140, 15, and <1% activity (k(cat)/K(m)) on starch, amylose DP17, and 2-chloro-4-nitrophenyl beta-d-maltoheptaoside, whereas T212Y at subsite +4 has 32, 370, and 90% activity, respectively. Thus engineering of aromatic stacking interactions at the ends of the 10-subsite long binding cleft affects activity very differently, dependent on the substrate. Y105A dominates in dual subsite -6/+4 [Y105A/T212(Y/W)]AMY1 mutants having almost retained and low activity on starch and oligosaccharides, respectively. Bond cleavage analysis of oligosaccharide degradation by wild-type and mutant AMY1 supports that Tyr(105) is critical for binding at subsite -6. Substrate binding is improved by T212(Y/W) introduced at subsite +4 and the [Y105A/T212(Y/W)]AMY1 double mutants synergistically enhanced productive binding of the substrate aglycone. The enzymatic properties of the series of AMY1 mutants suggest that longer substrates adopt several binding modes. This is in excellent agreement with computed distinct multiple docking solutions observed for maltododecaose at outer binding areas of AMY1 beyond subsites -3 and +3.     

Automated docking of alpha-(1-->4)- and alpha-(1-->6)-linked glucosyl trisaccharides and maltopentaose into the soybean beta-amylase active site

The Lamarckian genetic algorithm of AutoDock 3.0 was used to dock alpha-maltotriose, methyl alpha-panoside, methyl alpha-isopanoside, methyl alpha-isomaltotrioside, methyl alpha-(6(1)-alpha-glucopyranosyl)-maltoside, and alpha-maltopentaose into the closed and, except for alpha-maltopentaose, into the open conformation of the soybean beta-amylase active site. In the closed conformation, the hinged flap at the mouth of the active site closes over the substrate. The nonreducing end of alpha-maltotriose docks preferentially to subsites -2 or +1, the latter yielding nonproductive binding. Some ligands dock into less optimal conformations with the nonreducing end at subsite -1. The reducing-end glucosyl residue of nonproductively-bound alpha-maltotriose is close to residue Gln194, which likely contributes to binding to subsite +3. In the open conformation, the substrate hydrogen-bonds with several residues of the open flap. When the flap closes, the substrate productively docks if the nonreducing end is near subsites -2 or -1. Trisaccharides with alpha-(1-->6) bonds do not successfully dock except for methyl alpha-isopanoside, whose first and second glucosyl rings dock exceptionally well into subsites -2 and -1. The alpha-(1-->6) bond between the second and third glucosyl units causes the latter to be improperly positioned into subsite +1; the fact that isopanose is not a substrate of beta-amylase indicates that binding to this subsite is critical for hydrolysis.     

Structural basis for broad substrate specificity in higher plant beta-D-glucan glucohydrolases

Family 3 beta-D-glucan glucohydrolases are distributed widely in higher plants. The enzymes catalyze the hydrolytic removal of beta-D-glucosyl residues from nonreducing termini of a range of beta-D-glucans and beta-D-oligoglucosides. Their broad specificity can be explained by x-ray crystallographic data obtained from a barley beta-D-glucan glucohydrolase in complex with nonhydrolyzable S-glycoside substrate analogs and by molecular modeling of enzyme/substrate complexes. The glucosyl residue that occupies binding subsite -1 is locked tightly into a fixed position through extensive hydrogen bonding with six amino acid residues near the bottom of an active site pocket. In contrast, the glucosyl residue at subsite +1 is located between two Trp residues at the entrance of the pocket, where it is constrained less tightly. The relative flexibility of binding at subsite +1, coupled with the projection of the remainder of bound substrate away from the enzyme's surface, means that the overall active site can accommodate a range of substrates with variable spatial dispositions of adjacent beta-D-glucosyl residues. The broad specificity for glycosidic linkage type enables the enzyme to perform diverse functions during plant development.     

Engineering cyclodextrin glycosyltransferase into a starch hydrolase with a high exo-specificity

Cyclodextrin glycosyltransferase (CGTase) enzymes from various bacteria catalyze the formation of cyclodextrins from starch. The Bacillus stearothermophilus maltogenic alpha-amylase (G2-amylase is structurally very similar to CGTases, but converts starch into maltose. Comparison of the three-dimensional structures revealed two large differences in the substrate binding clefts. (i) The loop forming acceptor subsite +3 had a different conformation, providing the G2-amylase with more space at acceptor subsite +3, and (ii) the G2-amylase contained a five-residue amino acid insertion that hampers substrate binding at the donor subsites -3/-4 (Biochemistry, 38 (1999) 8385). In an attempt to change CGTase into an enzyme with the reaction and product specificity of the G2-amylase, which is used in the bakery industry, these differences were introduced into Thermoanerobacterium thermosulfurigenes CGTase. The loop forming acceptor subsite +3 was exchanged, which strongly reduced the cyclization activity, however, the product specificity was hardly altered. The five-residue insertion at the donor subsites drastically decreased the cyclization activity of CGTase to the extent that hydrolysis had become the main activity of enzyme. Moreover, this mutant produces linear products of variable sizes with a preference for maltose and had a strongly increased exo-specificity. Thus, CGTase can be changed into a starch hydrolase with a high exo-specificity by hampering substrate binding at the remote donor substrate binding subsites.     

Role of the loop structure of the catalytic domain in rice class I chitinase

In the three-dimensional structure of a rice class I chitinase (OsChia1b) determined recently, a loop structure (loop II) is located at the end of the substrate-binding cleft, and is thus suggested to be involved in substrate binding. In order to test this assumption, deletion of the loop II region from the catalytic domain of OsChia1b and replacement of Trp159 in loop II with Ala were carried out. The loop II deletion and the W159A mutation increased hydrolytic activity not only towards (GlcNAc)6 but also towards polysaccharide substrates. Similar results were obtained for kcat/Km values determined for substrate reduced-(GlcNAc)5. The two mutations shifted the splitting positions in (GlcNAc)6 to the reducing end side, but the shift was less intensive in the Trp mutant. Theoretical analysis of the reaction time course indicated that sugar residue affinity at the +3 subsite was reduced from -2 kcal/mol to +0.5 kcal/mol by loop II deletion. Reduced affinity at the +3 subsite might enhance the release of product fragments, resulting in higher turnover and higher enzymatic activities. Thus, we concluded that loop II is involved in sugar residue binding at the +3 subsite, but that Trp159 itself appears to contribute only partly to sugar residue interaction at the subsite.     

Effects of essential carbohydrate/aromatic stacking interaction with Tyr100 and Phe259 on substrate binding of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp. 1011

The stacking interaction between a tyrosine residue and the sugar ring at the catalytic subsite -1 is strictly conserved in the glycoside hydrolase family 13 enzymes. Replacing Tyr100 with leucine in cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. 1011 to prevent stacking significantly decreased all CGTase activities. The adjacent stacking interaction with both Phe183 and Phe259 onto the sugar ring at subsite +2 is essentially conserved among CGTases. F183L/F259L mutant CGTase affects donor substrate binding and/or acceptor binding during transglycosylation [Nakamura et al. (1994) Biochemistry 33, 9929-9936]. To elucidate the precise role of carbohydrate/aromatic stacking interaction at subsites -1 and +2 on the substrate binding of CGTases, we analyzed the X-ray structures of wild-type (2.0 A resolution), and Y100L (2.2 A resolution) and F183L/F259L mutant (1.9 A resolution) CGTases complexed with the inhibitor, acarbose. The refined structures revealed that acarbose molecules bound to the Y100L mutant moved from the active center toward the side chain of Tyr195, and the hydrogen bonding and hydrophobic interaction between acarbose and subsites significantly diminished. The position of pseudo-tetrasaccharide binding in the F183L/F259L mutant was closer to the non-reducing end, and the torsion angles of glycosidic linkages at subsites -1 to +1 on molecule 1 and subsites -2 to -1 on molecule 2 significantly changed compared with that of each molecule of wild-type-acarbose complex to adopt the structural change of subsite +2. These structural and biochemical data suggest that substrate binding in the active site of CGTase is critically affected by the carbohydrate/aromatic stacking interaction with Tyr100 at the catalytic subsite -1 and that this effect is likely a result of cooperation between Tyr100 and Phe259 through stacking interaction with substrate at subsite +2.     

Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

Subsite affinity maps of long substrate binding clefts in barley alpha-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3-12, revealed unfavorable binding energies at the internal subsites -3 and -5 and at subsites -8 and +3/+4 defining these subsites as binding barriers. Barley alpha-amylase 1 mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in alpha-amylases.     

Force calculations in automated docking: enzyme-substrate interactions in Fusarium oxysporum Cel7B

AutoDock is a small-molecule docking program that uses an energy function to score docked ligands. Here AutoDock's grid-based method for energy evaluation was exploited to evaluate the force exerted by Fusarium oxysporum Cel7B on the atoms of docked cellooligosaccharides and a thiooligosaccharide substrate analog. Coupled with the interaction energies evaluated for each docked ligand, these forces give insight into the dynamics of the ligand in the active site, and help to elucidate the relative importance of specific enzyme-substrate interactions in stabilizing the substrate transition-state conformation. The processive force on the docked substrate in the F. oxysporum Cel7B active site is less than half of that on the docked substrate in the Hypocrea jecorina Cel7A active site. Hydrogen bonding interactions of the enzyme with the C2 hydroxyl group of the glucosyl residue in subsite -2 and with the C3 hydroxyl group of the glucosyl residue in subsite +1 are the most significant in stabilizing the distorted14B transition-state conformation of the glucosyl residue in subsite -1. The force calculations also help to elucidate the mechanism that prevents the active site from fouling.     

Mechanism of chitosanase-oligosaccharide interaction: subsite structure of Streptomyces sp. N174 chitosanase and the role of Asp57 carboxylate.

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.     

Identification of acceptor substrate binding subsites +2 and +3 in the amylomaltase from Thermus thermophilus HB8

Glycoside hydrolase family 77 (GH77) belongs to the alpha-amylase superfamily (Clan H) together with GH13 and GH70. GH77 enzymes are amylomaltases or 4-alpha-glucanotransferases, involved in maltose metabolism in microorganisms and in starch biosynthesis in plants. Here we characterized the amylomaltase from the hyperthermophilic bacterium Thermus thermophilus HB8 (Tt AMase). Site-directed mutagenesis of the active site residues (Asp293, nucleophile; Glu340, general acid/base catalyst; Asp395, transition state stabilizer) shows that GH77 Tt AMase and GH13 enzymes share the same catalytic machinery. Quantification of the enzyme's transglycosylation and hydrolytic activities revealed that Tt AMase is among the most efficient 4-alpha-glucanotransferases in the alpha-amylase superfamily. The active site contains at least seven substrate binding sites, subsites -2 and +3 favoring substrate binding and subsites -3 and +2 not, in contrast to several GH13 enzymes in which subsite +2 contributes to oligosaccharide binding. A model of a maltoheptaose (G7) substrate bound to the enzyme was used to probe the details of the interactions of the substrate with the protein at acceptor subsites +2 and +3 by site-directed mutagenesis. Substitution of the fully conserved Asp249 with a Ser in subsite +2 reduced the activity 23-fold (for G7 as a substrate) to 385-fold (for maltotriose). Similar mutations reduced the activity of alpha-amylases only up to 10-fold. Thus, the characteristics of acceptor subsite +2 represent a main difference between GH13 amylases and GH77 amylomaltases.     

Dissecting the catalytic mechanism of a plant beta-D-glucan glucohydrolase through structural biology using inhibitors and substrate analogues

Higher plant, family GH3 beta-D-glucan glucohydrolases exhibit exo-hydrolytic and retaining (e-->e) mechanisms of action and catalyze the removal of single glucosyl residues from the non-reducing termini of beta-D-linked glucosidic substrates, with retention of anomeric configuration. The broad specificity beta-D-glucan glucohydrolases are likely to play roles in cell wall re-modelling, turn-over of cell wall components and possibly in plant defence reactions against pathogens. Crystal structures of the barley beta-D-glucan glucohydrolase, obtained from both native enzyme and from the enzyme in complex with a substrate analogues and mechanism-based inhibitors, have enabled the basis of substrate specificity, the mechanism of catalysis, and the role of domain movements during the catalytic cycle to be defined in precise molecular terms. The active site of the enzyme forms a shallow 'pocket' that is located at the interface of two domains of the enzyme and accommodates two glucosyl residues. The propensity of the enzyme to hydrolyze a broad range of substrates with (1-->2)-, (1-->3)-, (1-->4)- and (1-->6)-beta-D-glucosidic linkages is explained from crystal structures of the enzyme in complex with non-hydrolysable S-glycoside substrate analogues, and from molecular modelling. During binding of gluco-oligosaccharides, the glucosyl residue at subsite -1 is locked in a highly constrained position, but the glucosyl residue at the +1 subsite is free to adjust its position between two tryptophan residues positioned at the entry of the active site pocket. The flexibility at subsite +1 and the projection of the remainder of the substrate away from the pocket provide a structural rationale for the capacity of the enzyme to accommodate and hydrolyze glucosides with different linkage positions and hence different overall conformations. While mechanism-based inhibitors with micromolar Ki constants bind in the active site of the enzyme and form esters with the catalytic nucleophile, transition-state mimics bind with their 'glucose' moieties distorted into the 4E conformation, which is critical for the nanomolar binding of these inhibitors to the enzyme. The glucose product of the reaction, which is released from the non-reducing termini of substrates, remains bound to the beta-D-glucan glucohydrolase in the -1 subsite of the active site, until a new substrate molecule approaches the enzyme. If dissociation of the glucose from the enzyme active site could be synchronized throughout the crystal, time-resolved Laue X-ray crystallography could be used to follow the conformational changes that occur as the glucose product diffuses away and the incoming substrate is bound by the enzyme.     

Recognition of chitooligosaccharides and their N-acetyl groups by putative subsites of chitin deacetylase from a deuteromycete, Colletotrichum lindemuthianum

The reaction pattern of an extracellular chitin deacetylase from a Deuteromycete, Colletotrichum lindemuthianum ATCC 56676, was investigated by use of chitooligosaccharides [(GlcNAc)(n)(), n = 3-6] and partially N-deacetylated chitooligosaccharides as substrates. When 0.5% of (GlcNAc)(n)() was deacetylated, the corresponding monodeacetylated products were initially detected without any processivity, suggesting the involvement of a multiple-chain mechanism for the deacetylation reaction. The structural analysis of these first-step products indicated that the chitin deacetylase strongly recognizes a sequence of four N-acetyl-D-glucosamine (GlcNAc) residues of the substrate (the subsites for the four GlcNAc residues are defined as -2, -1, 0, and +1, respectively, from the nonreducing end to the reducing end), and the N-acetyl group in the GlcNAc residue positioned at subsite 0 is exclusively deacetylated. When substrates of a low concentration (100 microM) were deacetylated, the initial deacetylation rate for (GlcNAc)(4) was comparable to that of (GlcNAc)(5), while deacetylation of (GlcNAc)(3) could not be detected. Reaction rate analyses of partially N-deacetylated chitooligosaccharides suggested that subsite -2 strongly recognizes the N-acetyl group of the GlcNAc residue of the substrate, while the deacetylation rate was not affected when either subsite -1 or +1 was occupied with a D-glucosamine residue instead of GlcNAc residue. Thus, the reaction pattern of the chitin deacetylase is completely distinct from that of a Zygomycete, Mucor rouxii, which produces a chitin deacetylase for accumulation of chitosan in its cell wall.     

Mode of action of a family 75 chitosanase from Streptomyces avermitilis

Chitooligosaccharides (CHOS) are oligomers composed of glucosamine and N-acetylglucosamine with several interesting bioactivities that can be produced from enzymatic cleavage of chitosans. By controlling the degree of acetylation of the substrate chitosan, the enzyme, and the extent of enzyme degradation, CHOS preparations with limited variation in length and sequence can be produced. We here report on the degradation of chitosans with a novel family 75 chitosanase, SaCsn75A from Streptomyces avermitilis . By characterizing the CHOS preparations, we have obtained insight into the mode of action and subsite specificities of the enzyme. The degradation of a fully deacetylated and a 31% acetylated chitosan revealed that the enzyme degrade these substrates according to a nonprocessive, endo mode of action. With the 31% acetylated chitosan as substrate, the kinetics of the degradation showed an initial rapid phase, followed by a second slower phase. In the initial faster phase, an acetylated unit (A) is productively bound in subsite -1, whereas deacetylated units (D) are bound in the -2 subsite and the +1 subsite. In the slower second phase, D-units bind productively in the -1 subsite, probably with both acetylated and deacetylated units in the -2 subsite, but still with an absolute preference for deacetylated units in the +1 subsite. CHOS produced in the initial phase are composed of deacetylated units with an acetylated reducing end. In the slower second phase, higher amounts of low DP fully deacetylated oligomers (dimer and trimer) are produced, while the higher DP oligomers are dominated by compounds with acetylated reducing ends containing increasing amounts of internal acetylated units. The degradation of chitosans with varying degrees of acetylation to maximum extents of degradation showed that increasingly longer oligomers are produced with increasing degree of acetylation, and that the longer oligomers contain sequences of consecutive acetylated units interspaced by single deacetylated units. The catalytic properties of SaCsn75A differ from the properties of a previously characterized family 46 chitosanase from S. coelicolor (ScCsn46A).     

Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley limit dextrinase

Barley limit dextrinase (HvLD) of glycoside hydrolase family 13 is the sole enzyme hydrolysing α-1,6-glucosidic linkages from starch in the germinating seed. Surprisingly, HvLD shows 150- and 7-fold higher activity towards pullulan and β-limit dextrin, respectively, than amylopectin. This is investigated by mutational analysis of residues in the N-terminal CBM-21-like domain (Ser14Arg, His108Arg, Ser14Arg/His108Arg) and at the outer subsites +2 (Phe553Gly) and +3 (Phe620Ala, Asp621Ala, Phe620Ala/Asp621Ala) of the active site. The Ser14 and His108 mutants mimic natural LD variants from sorghum and rice with elevated enzymatic activity. Although situated about 40 Å from the active site, the single mutants had 15–40% catalytic efficiency compared to wild type for the three polysaccharides and the double mutant retained 27% activity for β-limit dextrin and 64% for pullulan and amylopectin. These three mutants hydrolysed 4,6-O-benzylidene-4-nitrophenyl-6³-α-d-maltotriosyl-maltotriose (BPNPG3G3) with 51–109% of wild-type activity. The results highlight that the N-terminal CBM21-like domain plays a role in activity. Phe553 and the highly conserved Trp512 sandwich a substrate main chain glucosyl residue at subsite +2 of the active site, while substrate contacts of Phe620 and Asp621 at subsite +3 are less prominent. Phe553Gly showed 47% and 25% activity on pullulan and BPNPG3G3, respectively having a main role at subsite +2. By contrast at subsite +3, Asp621Ala increased activity on pullulan by 2.4-fold, while Phe620Ala/Asp621Ala retained only 7% activity on pullulan albeit showed 25% activity towards BPNPG3G3. This outcome supports that the outer substrate binding area harbours preference determinants for the branched substrates amylopectin and β-limit dextrin.     

Involvement of individual subsites and secondary substrate binding sites in multiple attack on amylose by barley alpha-amylase

Barley alpha-amylase 1 (AMY1) hydrolyzed amylose with a degree of multiple attack (DMA) of 1.9; that is, on average, 2.9 glycoside bonds are cleaved per productive enzyme-substrate encounter. Six AMY1 mutants, spanning the substrate binding cleft from subsites -6 to +4, and a fusion protein, AMY1-SBD, of AMY1 and the starch binding domain (SBD) of Aspergillus niger glucoamylase were also analyzed. DMA of the subsite -6 mutant Y105A and AMY1-SBD increased to 3.3 and 3.0, respectively. M53E, M298S, and T212W at subsites -2, +1/+2, and +4, respectively, and the double mutant Y105A/T212W had decreased DMA of 1.0-1.4. C95A (subsite -5) had a DMA similar to that of wild type. Maltoheptaose (G7) was always the major initial oligosaccharide product. Wild-type and the subsite mutants released G6 at 27-40%, G8 at 60-70%, G9 at 39-48%, and G10 at 33-44% of the G7 rate, whereas AMY1-SBD more efficiently produced G8, G9, and G10 at rates similar to, 66%, and 60% of G7, respectively. In contrast, the shorter products appeared with large individual differences: G1, 0-15%; G2, 8-43%; G3, 0-22%; and G4, 0-11% of the G7 rate. G5 was always a minor product. Multiple attack thus involves both longer translocation of substrate in the binding cleft upon the initial cleavage to produce G6-G10, essentially independent of subsite mutations, and short-distance moves resulting in individually very different rates of release of G1-G4. Accordingly, the degree of multiple attack as well as the profile of products can be manipulated by structural changes in the active site or by introduction of extra substrate binding sites.