Kinetic characterization of Aspergillus niger N400 endopolygalacturonases I, II and C.

Endopolygalacturonases I, II and C isolated from recombinant Aspergillus niger strains were characterized with respect to pH optimum, activity on polygalacturonic acid and mode of action and kinetics on oligogalacturonates of different chain length (n = 3-7). Apparent Vmax values using polygalacturonate as a substrate at the pH optimum, pH 4.1, were calculated as 13.8 mukat.mg-1, 36.5 mukat.mg-1 and 415 nkat.mg-1 for endopolygalacturonases I, II and C, respectively. K(m) values were < 0.15 mg.mL-1 for all three enzymes. Product progression analysis using polygalacturonate as a substrate revealed a random cleavage pattern for all three enzymes and suggested processive behavior for endopolygalacturonases I and C. This result was confirmed by analysis of the mode of action using oligogalacturonates. Processivity was observed when the degree of polymerization of the substrate exceeded 5 or 6 for endopolygalacturonase I and endopolygalacturonase C, respectively. The bond-cleavage frequencies obtained for the hydrolysis of the oligogalacturonates were used to assess subsite maps. The maps indicate that the minimum number of subsites is seven for all three enzymes. Using pectins of various degrees of esterification, it was shown that endopolygalacturonase II is the most sensitive to the presence of methyl esters. Like endopolygalacturonase II, endopolygalacturonases I, C and E, which was also included in this part of the study, preferred the non-esterified pectate. Additional differences in substrate specificity were revealed by analysis of the reaction products of hydrolysis of a mixture of pectate lyase-generated delta 4,5-unsaturated oligogalacturonates of degree of polymerization 4-8. Whereas endopolygalacturonase I showed a strong preference for generating the delta 4,5-unsaturated dimer, with endopolygalacturonase II the delta 4,5-unsaturated trimer accumulated, indicating further differences in substrate specificity. For endopolygalacturonases C and E both the delta 4,5-unsaturated dimer and trimer were observed, although in different ratios.     

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.     

The active site topology of Aspergillus niger endopolygalacturonase II as studied by site-directed mutagenesis

Strictly conserved charged residues among polygalacturonases (Asp-180, Asp-201, Asp-202, His-223, Arg-256, and Lys-258) were subjected to site-directed mutagenesis in Aspergillus niger endopolygalacturonase II. Specific activity, product progression, and kinetic parameters (K(m) and V(max)) were determined on polygalacturonic acid for the purified mutated enzymes, and bond cleavage frequencies on oligogalacturonates were calculated. Depending on their specific activity, the mutated endopolygalacturonases II were grouped into three classes. The mutant enzymes displayed bond cleavage frequencies on penta- and/or hexagalacturonate different from the wild type endopolygalacturonase II. Based on the biochemical characterization of endopolygalacturonase II mutants together with the three-dimensional structure of the wild type enzyme, we suggest that the mutated residues are involved in either primarily substrate binding (Arg-256 and Lys-258) or maintaining the proper ionization state of a catalytic residue (His-223). The individual roles of Asp-180, Asp-201, and Asp-202 in catalysis are discussed. The active site topology is different from the one commonly found in inverting glycosyl hydrolases.     

Performance of selected microbial pectinases on synthetic monomethyl-esterified di- and trigalacturonates

Two monomethyl esters of alpha-(1-4)-linked D-galacturonic dimers and three monomethyl esters of alpha-(1-4)-linked D-galacturonic acid trimers were synthesized chemically and further used as substrates in order to establish the substrate specificity of six different endopolygalacturonases from Aspergillus niger, one exopolygalacturonase from Aspergillus tubingensis, and four selected Erwinia chrysanthemi pectinases; exopolygalacturonan hydrolase X (PehX), exopolygalacturonate lyase X (PelX), exopectate lyase W (PelW), and oligogalacturonan lyase (Ogl). All A. niger endopolygalacturonases (PGs) were unable to hydrolyze the two monomethyldigalacturonates and 2-methyltrigalacturonate, whereas 1-methyltrigalacturonate was only cleaved by PGI, PGII, and PGB albeit at an extremely low rate. The hydrolysis of 3-methyltrigalacturonate into 2-methyldigalacturonate and galacturonate by all endopolygalacturonases demonstrates that these enzymes can accommodate a methylgalacturonate at subsite -2. The A. tubingensis exopolygalacturonase hydrolyzed the monomethyl-esterified digalacturonates and trigalacturonates although at lower rates than for the corresponding oligogalacturonates. 1-Methyltrigalacturonate was hydrolyzed at the same rate as trigalacturonate which demonstrates that the presence of a methyl ester at the third galacturonic acid from the nonreducing end does not have any effect on the performance of exopolygalacturonase. Of the four E. chrysanthemi pectinases, Ogl was the only enzyme able to cleave digalacturonate, whereas all four enzymes cleaved trigalacturonate. Ogl does not cleave monomethyl-esterified digalacturonate and trigalacturonate in case the second galacturonic acid residue from the reducing end is methyl-esterified. PehX did not hydrolyze any of the monomethyl-esterified trigalacturonates. The two lyases, PelX and PelW, were both only able to cleave 1-methyltrigalacturonate into Delta4,5-unsaturated 1-methyldigalacturonate and galacturonate.     

Active-site architecture of endopolygalacturonase I from Stereum purpureum revealed by crystal structures in native and ligand-bound forms at atomic resolution

Crystal structures of endopolygalacturonase from Stereum purpureum were solved in native and two galacturonic acid complex states at atomic resolution. Endopolygalacturonase catalyzes the hydrolysis of alpha-1,4-glycosidic linkage of polygalacturonate in pectin. The native structure was determined by the multiple wavelength anomalous dispersion method and was refined anisotropically with SHELXL-97, with an R factor of 11.4% and an R(free) factor of 14.0% at 0.96 A resolution. The enzyme folds into a right-handed parallel beta-helix with 10 complete turns. The crystal structures of its binary complex with one D-galacturonate and its ternary complex with two D-galacturonates were also determined to identify the substrate binding site at 1.0 and 1.15 A resolutions, respectively. In the binary complex, one beta-D-galactopyranuronate was found in the +1 subsite, thus proving the strong affinity of the +1 subsite expected from the bond cleavage frequency on oligogalacturonates. In the ternary complex, an additional beta-D-galactofuranuronate was found in the -1 subsite. In both subsites, the recognition of the galacturonate carboxy group is important in galacturonate binding. In the +1 subsite, the carboxy group interacts with three basic residues, His195, Arg226, and Lys228, which were conserved in all endopolygalacturonases. In the -1 subsite, the unique nonprolyl cis-peptide bond is believed to be involved in binding the carboxy group of the substrate. The active site architecture of the complexes provides insight into the mechanism of inverting glycosyl hydrolases and also sheds light on the basis of the differences between the family 28 and the other inverting glycosyl hydrolases.     

Structural insights into the processivity of endopolygalacturonase I from Aspergillus niger

Endopolygalacturonase I is a processive enzyme, while the 60% sequence identical endopolygalacturonase II is not. The 1.70 A resolution crystal structure of endopolygalacturonase I reveals a narrowed substrate binding cleft. In addition, Arg96, a residue in this cleft previously shown to be critical for processivity, interacts with the substrate mimics glycerol and sulfate in several well-defined conformations in the six molecules in the asymmetric unit. From this we conclude that both Arg96 and the narrowed substrate binding cleft contribute to retaining the substrate while it moves through the active site after a cleavage event has occurred.     

Characterization of a novel endopolygalacturonase from Aspergillus niger with unique kinetic properties

We isolated and characterized a new type of endopolygalacturonase (PG)-encoding gene, pgaD, from Aspergillus niger. The primary structure of PGD differs from that of other A. niger PGs by a 136 amino acid residues long N-terminal extension. Biochemical analysis demonstrated extreme processive behavior of the enzyme on oligomers longer than five galacturonate units. Furthermore, PGD is the only A. niger PG capable of hydrolyzing di-galacturonate. It is tentatively concluded that the enzyme is composed of four subsites. The physiological role of PGD is discussed.     

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.     

Functional and structural effects of mutagenic replacement of Asn37 at subsite F on the lysozyme-catalyzed reaction

To investigate the functional role of subsites E and F in lysozyme catalysis, Asn37 of hen egg-white lysozyme (HEL), which is postulated to participate in sugar residue binding at the right-sided subsite F through hydrogen bonding, was replaced by Ser or Gly by site-directed mutagenesis. The mutations of Asn37 neither significantly affected the binding constant for chitotriose nor the enzymatic activity toward the substrate glycol chitin. However, kinetic analysis with the substrate N-acetylglucosamine pentamer, (GlcNAc)(5), revealed that the conversion of Asn37 to Gly decreased the binding free energies for subsites E and F, while the conversion to Ser increased the substrate affinity at subsite F. It was further found that the rate constant of transglycosylation was reduced by these mutations. These results suggest that Asn37 is involved not only in substrate binding at subsite F but also in transglycosylation activity. No remarkable change in the tertiary structure except the side chain of the 37th residue was detected on X-ray analysis of the mutant proteins, indicating that the alterations in the enzymatic function between the wild type and mutant enzymes depend on limited structural change around the substitution site. It is thus speculated that the slight conformational difference in the side chain of position 37 may affect the substrate and acceptor binding at subsites E and F, leading to lower the efficiency of the transglycosylation activities of the mutant proteins.     

Chemicomechanical transduction performed by enzymes activated by polymers

Kinetic models for the mode of action of processive and non-processive DNA-helicases are detailed. Fluxes at the steady state are analyzed, and the random walk of the enzymes on the DNA is studied in connection with the rate constants of the chemical reactions involved in the transformation of substrate to products. Finally, the constants of the kinetic model for the processive helicase are related to the parameters of an analogous viscoelastic model.     

Processive phosphorylation: mechanism and biological importance

Recent proteomic data indicate that a majority of the phosphorylated proteins in a eucaryotic cell contain multiple sites of phosphorylation. In many signaling events, a single kinase phosphorylates multiple sites on a target protein. Processive phosphorylation occurs when a protein kinase binds once to a substrate and phosphorylates all of the available sites before dissociating. In this review, we discuss examples of processive phosphorylation by serine/threonine kinases and tyrosine kinases. We describe current experimental approaches for distinguishing processive from non-processive phosphorylation. Finally, we contrast the biological situations that are suited to regulation by processive and non-processive phosphorylation.     

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.     

A Naturally Variable Residue in the S1 Subsite of M1 Family Aminopeptidases Modulates Catalytic Properties and Promotes Functional Specialization

M1 family metallo-aminopeptidases fulfill a wide range of critical and in some cases medically relevant roles in humans and human pathogens. The specificity of M1-aminopeptidases is dominated by the interaction of the well defined S1 subsite with the side chain of the first (P1) residue of the substrate and can vary widely. Extensive natural variation occurs at one of the residues that contributes to formation of the cylindrical S1 subsite. We investigated whether this natural variation contributes to diversity in S1 subsite specificity. Effects of 11 substitutions of the S1 subsite residue valine 459 in the Plasmodium falciparum aminopeptidase PfA-M1 and of three substitutions of the homologous residue methionine 260 in Escherichia coli aminopeptidase N were characterized. Many of these substitutions altered steady-state kinetic parameters for dipeptide hydrolysis and remodeled S1 subsite specificity. The most dramatic change in specificity resulted from substitution with proline, which collapsed S1 subsite specificity such that only substrates with P1-Arg, -Lys, or -Met were appreciably hydrolyzed. The structure of PfA-M1 V459P revealed that the proline substitution induced a local conformational change in the polypeptide backbone that resulted in a narrowed S1 subsite. The restricted specificity and active site backbone conformation of PfA-M1 V459P mirrored those of endoplasmic reticulum aminopeptidase 2, a human enzyme with proline in the variable S1 subsite position. Our results provide compelling evidence that changes in the variable residue in the S1 subsite of M1-aminopeptidases have facilitated the evolution of new specificities and ultimately novel functions for this important class of enzymes.     

The active site of an acidic endo-1,4-beta-xylanase of Aspergillus niger

The substrate binding site of an acidic endo-1,4-beta-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) of Aspergillus niger was investigated using 1,4-beta-xylooligosaccharides (1-3H)-labelled at the reducing end. Bond cleavage frequencies and V/Km parameters of the oligosaccharides were determined under conditions of unimolecular hydrolysis and, according to the method of Suganuma et al. (J. Biochem. (Tokyo) (1978) 84, 293-316), used for evaluation of subsite affinities. The substrate binding site of the enzyme was found to consist of seven subsites, numbered -IV, -III, -II, -I, I, II and III, towards the subsite binding the reducing end unit of xyloheptaose. The catalytic groups were localized between subsites -I and I, the affinities of which have not been determined. All other subsites showed positive values of affinities for binding xylosyl residues. The values decrease from subsites -II and II, similarly in both directions. As a consequence of such an almost symmetric distribution of affinities around the catalytic groups, the enzyme cleaves preferentially the bonds in the oligosaccharides which are most distant from both terminals. Thus, the acidic A. niger beta-xylanase appears to be an endo-1,4-beta-xylanase attacking polymeric substrates in a random fashion. This conclusion was supported by viscosimetric measurements with carboxymethylxylan as a substrate.     

Comparative studies of chitinases A, B and C from Serratia marcescens

Serratia marcescens produces three chitinases, ChiA, ChiB and ChiC which together enable the bacterium to efficiently degrade the insoluble chitin polymer. We present an overview of the structural properties of these enzymes, as well as an analysis of their activities towards artificial chromogenic chito-oligosaccharide-based substrates, chito-oligosaccharides, chitin and chitosan. We also present comparative inhibition data for the pseudotrisaccharide allosamidin (an analogue of the reaction intermediate) and the cyclic pentapeptide argadin. The results show that the enzymes differ in terms of their subsite architecture and their efficiency towards chitinous substrates. The idea that the three chitinases play different roles during chitin degradation was confirmed by the synergistic effects that were observed for certain combinations of the enzymes. Studies of the degradation of the soluble heteropolymer chitosan provided insight into processivity. Taken together, the available data for Serratia chitinases show that the chitinolytic machinery of this bacterium consists of two processive exo-enzymes that degrade the chitin chains in opposite directions (ChiA and ChiB) and a non-processive endo-enzyme, ChiC.     

Val326 of Thermoactinomyces vulgaris R-47 amylase II modulates the preference for alpha-(1,4)- and alpha-(1,6)-glycosidic linkages

Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II) catalyzes not only the hydrolysis of alpha-(1,4)- and alpha-(1,6)-glycosidic linkages but also transglycosylation. The subsite +1 structure of alpha-amylase family enzymes plays important roles in substrate specificity and transglycosylation activity. We focused on the amino acid residue at the 326th position based on information on the primary structure and crystal structure, and replaced Val with Ala, Ile, or Thr. The V326A mutant favored hydrolysis of the alpha-(1,4)-glycosidic linkage compared to the wild-type enzyme. In contrast, the V326I mutant favored hydrolysis of the alpha-(1,6)-glycosidic linkage and exhibited low transglycosylation activity. In the case of the V326T mutant, the hydrolytic activity was almost identical to that of the wild-type TVA II, and the transglycosylation activity was poor. These results suggest that the volume and the hydrophobicity of the amino acid residue at the 326th position modulate both the preference for glycosidic linkages and the transglycosylation activity.     

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.     

Substrate positioning in chitinase A, a processive chito-biohydrolase from Serratia marcescens

The contributions of the -3 subsite and a putative +3 subsite to substrate positioning in ChiA from Serratia marcescens have been investigated by comparing how ChiA and its -3 subsite mutant W167A interact with soluble substrates. The data show that Trp - GlcNAc stacking in the -3 subsite rigidifies the protein backbone supporting the formation of the intermolecular interaction network that is necessary for the recognition and positioning of the N-acetyl groups before the -1 subsite. The +3 subsite exhibits considerable substrate affinity that may promote endo-activity in ChiA and/or assist in expelling dimeric products from the +1 and +2 subsites during processive hydrolysis.     

Comparative studies of chitinases A,B and C from Serratia marcescens

Serratia marcescens produces three chitinases, ChiA, ChiB and ChiC which together enable the bacterium to efficiently degrade the insoluble chitin polymer. We present an overview of the structural properties of these enzymes, as well as an analysis of their activities towards artificial chromogenic chito-oligosaccharide-based substrates, chito-oligosaccharides, chitin and chitosan. We also present comparative inhibition data for the pseudotrisaccharide allosamidin (an analogue of the reaction intermediate) and the cyclic pentapeptide argadin. The results show that the enzymes differ in terms of their subsite architecture and their efficiency towards chitinous substrates. The idea that the three chitinases play different roles during chitin degradation was confirmed by the synergistic effects that were observed for certain combinations of the enzymes. Studies of the degradation of the soluble heteropolymer chitosan provided insight into processivity. Taken together, the available data for Serratia chitinases show that the chitinolytic machinery of this bacterium consists of two processive exo-enzymes that degrade the chitin chains in opposite directions (ChiA and ChiB) and a non-processive endo-enzyme, ChiC.