Characterization of an endo-beta-1,6-Galactanase from Streptomyces avermitilis NBRC14893

The putative endo-beta-1,6-galactanase gene from Streptomyces avermitilis was cloned and expressed in Escherichia coli, and the enzymatic properties of the recombinant enzyme were characterized. The gene consisted of a 1,476-bp open reading frame and encoded a 491-amino-acid protein, comprising an N-terminal secretion signal sequence and glycoside hydrolase family 5 catalytic module. The recombinant enzyme, Sa1,6Gal5A, catalyzed the hydrolysis of beta-1,6-linked galactosyl linkages of oligosaccharides and polysaccharides. The enzyme produced galactose and a range of beta-1,6-linked galacto-oligosaccharides, predominantly beta-1,6-galactobiose, from beta-1,6-galactan chains. There was a synergistic effect between the enzyme and Sa1,3Gal43A in degrading tomato arabinogalactan proteins. These results suggest that Sa1,6Gal5A is the first identified endo-beta-1,6-galactanase from a prokaryote.     

Structural basis for the substrate specificity of a Bacillus 1,3-1,4-beta-glucanase

Depolymerization of polysaccharides is catalyzed by highly specific enzymes that promote hydrolysis of the scissile glycosidic bond by an activated water molecule. 1,3-1,4-beta-Glucanases selectively cleave beta-1,4 glycosidic bonds in 3-O-substituted glucopyranosyl units within polysaccharides with mixed linkage. The reaction follows a double-displacement mechanism by which the configuration of the anomeric C(1)-atom of the glucosyl unit in subsite -I is retained. Here we report the high-resolution crystal structure of the hybrid 1,3-1,4-beta-glucanase H(A16-M)(E105Q/E109Q) in complex with a beta-glucan tetrasaccharide. The structure shows four beta-d-glucosyl moieties bound to the substrate-binding cleft covering subsites -IV to -I, thus corresponding to the reaction product. The ten active-site residues Asn26, Glu63, Arg65, Phe92, Tyr94, Glu105, Asp107, Glu109, Asn182 and Trp184 form a network of hydrogen bonds and hydrophobic stacking interactions with the substrate. These residues were previously identified by mutational analysis as significant for stabilization of the enzyme-carbohydrate complex, with Glu105 and Glu109 being the catalytic residues. Compared to the Michaelis complex model, the tetrasaccharide moiety is slightly shifted toward that part of the cleft binding the non-reducing end of the substrate, but shows previously unanticipated strong stacking interactions with Phe92 in subsite -I. A number of specific hydrogen-bond contacts between the enzyme and the equatorial O(2), O(3) and O(6) hydroxyl groups of the glucosyl residues in subsites -I, -II and -III are the structural basis for the observed substrate specificity of 1,3-1,4-beta-glucanases. Kinetic analysis of enzyme variants with the all beta-1,3 linked polysaccharide laminarin identified key residues mediating substrate specificity in good agreement with the structural data. The comparison with structures of the apo-enzyme H(A16-M) and a covalent enzyme-inhibitor (E.I) complex, together with kinetic and mutagenesis data, yields new insights into the structural requirements for substrate binding and catalysis. A detailed view of enzyme-carbohydrate interactions is presented and mechanistic implications are discussed.     

Crystal structure of truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase in complex with beta-1,3-1,4-cellotriose

Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (Fsbeta-glucanase) catalyzes the specific hydrolysis of beta-1,4 glycosidic bonds adjacent to beta-1,3 linkages in beta-D-glucans or lichenan. This is the first report to elucidate the crystal structure of a truncated Fsbeta-glucanase (TFsbeta-glucanase) in complex with beta-1,3-1,4-cellotriose, a major product of the enzyme reaction. The crystal structures, at a resolution of 2.3 angstroms, reveal that the overall fold of TFsbeta-glucanase remains virtually unchanged upon sugar binding. The enzyme accommodates five glucose residues, forming a concave active cleft. The beta-1,3-1,4-cellotriose with subsites -3 to -1 bound to the active cleft of TFsbeta-glucanase with its reducing end subsite -1 close to the key catalytic residues Glu56 and Glu60. All three subsites of the beta-1,3-1,4-cellotriose adopted a relaxed C(1)4 conformation, with a beta-1,3 glycosidic linkage between subsites -2 and -1, and a beta-1,4 glycosidic linkage between subsites -3 and -2. On the basis of the enzyme-product complex structure observed in this study, a catalytic mechanism and substrate binding conformation of the active site of TFsbeta-glucanase is proposed.     

Computational analysis of glycoside hydrolase family 1 specificities

Glycoside hydrolase family 1 consists of beta-glucosidases, beta-galactosidases, 6-phospho-beta-galactosidases, myrosinases, and other enzymes having similar primary and tertiary structures but diverse specificities. Among these enzymes, beta-glucosidases hydrolyze cellobiose to glucose, and therefore they are key players in any cellulose to glucose process. All family members attack beta-glycosidic bonds between a pyranosyl glycon and an aglycon, but most have little specificity for the aglycon or for the bond configuration. Furthermore, glycon specificity is not absolute. Sixteen family members (six beta-glucosidases, two cyanogenic beta-glucosidases, one 6-phospho-beta-galactosidase, two myrosinases, and five beta-glycosidases) have known tertiary structures. We have used automated docking to computationally bind disaccharides with allopyranosyl, galactopyranosyl, glucopyranosyl, mannopyranosyl, 6-phosphogalactopyranosyl, and 6-phosphoglucopyranosyl glycons, all linked by beta-(1,2), beta-(1,3), beta-(1,4), and beta-(1,6)-glycosidic bonds to beta-glucopyranoside aglycons, along with beta-(1,1-thio)-allopyranosyl, -galactopyranosyl, -glucopyranosyl, and -mannopyranosyl) beta-glucopyranosides, into all of these structures to investigate the structural determinants of their enzyme specificities. The following are the eight active-site residues: Glu191, Thr194, Phe205, Asn285, Arg336, Asn376, Trp378, and Trp465 (Zea mays beta-glucosidase numbering), that control a significant amount of glycon specificity. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 1021-1031, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Mutations of endo-beta-N-acetylglucosaminidase H active site residueAs sp130 anG glu132: activities and conformations

Endo-beta-N-acetylglucosaminidase H hydrolyzes the beta-(1-4)-glycosidic link of the N,N'-diacetylchitobiose core of high-mannose and hybrid asparagine-linked oligosaccharides. Seven mutants of the active site residues, Asp130 and Glu132, have been prepared, assayed, and crystallized. They include single site mutants of each residue to the corresponding amide, to Ala and to the alternate acidic residue, and to the double amide mutant. The mutants of Asp130 are more active than the corresponding Glu132 mutants, consistent with the assignment of the latter residue as the primary catalytic residue. The amide mutants are more active than the alternate acidic residue mutants, which in turn are more active than the Ala mutants. The structures of the Asn mutant of Asp130 and the double mutant are very similar to that of the wild-type enzyme. Several residues surrounding the mutated residues, including some that form part of the core of the beta-barrel and especially Tyr168 and Tyr244, adopt a very different conformation in the structures of the other two mutants of Asp130 and in the Asp mutant of Glu132. The results show that the residues in the upper layers of the beta-barrel can organize into two very distinct packing arrangements that depend on subtle electrostatic and steric differences and that greatly affect the geometry of the substrate-binding cleft. Consequently, the relative activities of several of the mutants are defined by structural changes, leading to impaired substrate binding, in addition to changes in functionality.     

Substrate recognition mechanism of alpha-1,6-glucosidic linkage hydrolyzing enzyme, dextran glucosidase from Streptococcus mutans

We have determined the crystal structure of Streptococcus mutans dextran glucosidase, which hydrolyzes the α-1,6-glucosidic linkage of isomaltooligosaccharides from their non-reducing ends to produce α-glucose. By using the mutant of catalytic acid Glu236→Gln, its complex structure with the isomaltotriose, a natural substrate of this enzyme, has been determined. The enzyme has 536 amino acid residues and a molecular mass of 62,001 Da. The native and the complex structures were determined by the molecular replacement method and refined to 2.2 Å resolution, resulting in a final R-factor of 18.3% for significant reflections in the native structure and 18.4% in the complex structure. The enzyme is composed of three domains, A, B and C, and has a (β/α)₈-barrel in domain A, which is common to the α-amylase family enzymes. Three catalytic residues are located at the bottom of the active site pocket and the bound isomaltotriose occupies subsites −1 to +2. The environment of the glucose residue at subsite −1 is similar to the environment of this residue in the α-amylase family. Hydrogen bonds between Asp60 and Arg398 and O4 atom of the glucose unit at subsite −1 accomplish recognition of the non-reducing end of the bound substrate. The side-chain atoms of Glu371 and Lys275 form hydrogen bonds with the O2 and O3 atoms of the glucose residue at subsite +1. The positions of atoms that compose the scissile α-1,6-glucosidic linkage (C1, O6 and C6 atoms) are identical with the positions of the atoms in the scissile α-1,4 linkage (C1, O4 and C4 atoms) of maltopentaose in the α-amylase structure from Bacillus subtilis. The comparison with the α-amylase suggests that Val195 of the dextran glucosidase and the corresponding residues of α-1,6-hydrolyzing enzymes participate in the determination of the substrate specificity of these enzymes.     

Purification and properties of a β-1,6-glucanase from Streptomyces sp. EF-14, an actinomycete antagonistic to Phytophthora spp.

Extracellular enzymes with glucanase activities are an important component of actinomycete-fungus antagonism. Streptomyces sp. EF-14 has been previously identified as one of the most potent antagonists of Phytophthora spp. A beta-1,6-glucanase (EC 3.2.1.75; glucan endo-1,6-beta-glucosidase) was purified by four chromatographic steps from the culture supernatant of strain EF-14 grown on a medium with lyophilized cells of Candida utilis as main nutrient source. The glucanase level in this medium followed a characteristic pattern in which the rise of beta-1,6-glucanase activity always preceded that of beta-1,3-glucanase. The molecular mass of the enzyme was estimated to be 65 kDa and the pI approximately 5.5. It hydrolyzed pustulan by an endo-mechanism generating gentiobiose and glucose as final products. Laminarin was not hydrolyzed indicating that the enzyme does not recognize beta-1,6-links flanked by beta-1,3-links. No significant clearing of yeast cell walls in liquid suspensions or in agar plates was observed indicating that this beta-1,6-glucanase is a non-lytic enzyme. This is the first beta-1,6-glucanase characterized from an actinomycete.     

Some thaumatin-like proteins hydrolyse polymeric β-1,3-glucans

Thaumatin and 12 purified thaumatin-like (TL) proteins were surveyed for their capacity to hydrolyse beta-1,3-glucans by using an in-gel glucanase assay. Six TL proteins identified by N-terminal amino acid microsequencing were found to be active on carboxymethyl(CM)-pachyman: a barley leaf stress-related permatin, two tomato fruit osmotins, a cherry fruit and two tobacco stigma proteins. TL enzymes ranged in specific activity from 0.07 to 89 nkat mg-1 with CM-pachyman as substrate. Hydrolytic activities were not restricted to TL proteins strongly binding to water-insoluble beta-1,3-glucans since the two osmotins were active without tight binding to pachyman. Some TL proteins hydrolysed crude fungal walls and one barley TL enzyme even lysed fungal spores. No activity was observed on laminarin in the in-gel hydrolase assay. Thin-layer chromatography revealed that the six enzymes acted as endo-beta-1, 3-glucanases leading to the formation of various oligoglucosides. Thus far, the TL enzymes (EC 3.2.1.x) appeared different from the well-known beta-1,3-glucanases (EC 3.2.1.39). No activity was found with thaumatin, zeamatin, tobacco leaf PR-R protein and four stress-related TL proteins from barley and pea. This is the first demonstration that diverse TL proteins are enzymatically active. The functions of some TL proteins must be reassessed because they display endo-beta-1,3-glucanase activity on polymeric beta-1, 3-glucans.     

Enzymes of the yeast lytic system produced by Arthrobacter GJM-1 bacterium and their role in the lysis of yeast cell walls.

Yeast lytic system produced by Arthrobacter GJM-1 bacterium during growth on baker's yeast cell walls contains a complete set of enzymes which can hydrolyze all structural components of cell walls of Saccharomyces cerevisiae. Chromatographic fractionation of the lytic system showed the presence of two types of endo-beta-1,3-glucanase. Rapid lysis of isolated cell walls of yeast was induced only by endo-beta-1,3-glucanase exhibiting high affinity to insoluble beta-1,3-glucans and releasing laminaripentaose as the main product of hydrolysis of beta-1,3-glucans. This enzyme was able to lyse intact cells of S. cerevisiae only in the presence of an additional factor present in the Arthrobacter GJM-1 lytic system, which was identified as an alkaline protease. This enzyme possesses the lowest molecular weight among other identified enzyme components present in the lytic system. Its role in the solubilization of yeast cell walls from the outer surface by endo-beta-1,3-glucanase could be substituted by preincubation of cells with Pronase or by allowing the glucanase to act on cells in the presence of thiol reagents. The mechanism of lysis of intact cells and isolated cell walls by the enzymes of Arthrobacter GJM-1 is discussed in the light of the present conception of yeast cell wall structure.     

Role of Ser216 in the mechanism of action of membrane-bound lytic transglycosylase B: further evidence for substrate-assisted catalysis

Lytic transglycosylases cleave the beta-(1-->4)-glycosidic bond in the bacterial cell wall heteropolymer peptidoglycan between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues with the concomitant formation of a 1,6-anhydromuramoyl residue. Based on sequence alignments, Ser216 in Pseudomonas aeruginosa membrane-bound lytic transglycosylase B (MltB) was targeted for replacement with alanine to delineate its role in the enzyme's mechanism of action. The specific activity of the Ser216-->Ala MltB derivative was less than 12% of that for the wild-type enzyme, while its substrate binding affinity remained virtually unaltered. These data are in agreement with a role of Ser216 in orienting the N-acetyl group on MurNAc at the -1 subsite of MltB for its participation in a substrate-assisted mechanism of action.     

Purification and properties of a beta-1,6-glucanase from Penicillium brefeldianum.

An inducible endo-beta-1,6-glucanase was purified from Penicillium brefeldianum by DEAE-cellulose, Bio-Gel P-150 and high-pressure liquid chromatography. The final preparation was essentially free from beta-1,3-glucanase and beta-glucosidase activities. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed one protein band with an Mr of 44000. The Vmax. and Km values were calculated to be 624 units (mumol/min)/mg and 2.78 mg/ml respectively. The glucanase had lytic activity against mycelial cells of the yeast Candida albicans. The yield of purified beta-1,6-glucanase from 100 mg dry weight of freeze-dried culture filtrate varied from 60 to 180 units.     

Cloning and expression of a novel Trichoderma viride laminarinase AI gene (lamAI)

The gene lamAI, which encodes a novel laminarinase AI of Trichoderma viride U-1, was cloned using RT-PCR in conjunction with the rapid amplification of cDNA ends (RACE) technique. The open reading frame consisted of 2,277 bp encoding a protein of 759 amino acid residues, including a 32-residue signal prepropeptide. The protein showed 91% sequence similarity to the putative Trichoderma virens beta-1,3-glucanase BGN1, but no significant similarity to fungal beta-1,6-glucanases or beta-1,3-glucanases from other organisms. On 40 h incubation with a solo carbon source, northern analysis revealed that the gene was induced by 0.5% laminaran from Eisenia bicyclis but was not by the same concentration of glucose. The lamAI cDNA was functionally expressed in the methylotrophic yeast Pichia pastoris, resulting in a recombinant enzyme with as high activity against laminaran as native LAMAI. Based on these data, the probable existence of endo-beta-1,3:1,6-glucan hydrolases as a subclass of endo-beta-1,3-glucanases in some mycoparasitic fungi is suggested.     

Role of arginine residues in the active site of the membrane-bound lytic transglycosylase B from Pseudomonas aeruginosa

Lytic transglycosylases cleave the beta-(1-->4)-glycosidic bond in the bacterial cell wall heteropolymer peptidoglycan between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues with the concomitant formation of a 1,6-anhydromuramoyl residue. On the basis of both sequence alignments with and structural considerations of soluble lytic transglycosylase Slt35 from Escherichia coli, four residues were predicted to be involved in substrate binding at the -1 subsite in the soluble derivative of Pseudomonas aeruginosa membrane-bound lytic transglycosylase MltB. These residues were targeted for site-specific replacement, and the effect on substrate binding and catalysis was determined. The residues Arg187 and Arg188, believed to be involved in binding the stem peptide on MurNAc, were shown to play an important role in substrate binding, as evidenced by peptidoglycan affinity assays and SUPREX analysis using MurNAc-dipeptide as ligand. The Michaelis-Menten parameters were determined for the respective mutants using insoluble peptidoglycan as substrate. In addition to affecting the steady-state binding of ligand to enzyme, as indicated by increases in K(M) values, significant decreases in k(cat) values suggested that replacement of either Arg187 and Arg188 with alanine perturbed the stabilization of both the transition state(s) and reaction intermediate. Thus, it appears that Arg187 and Arg188 are vital for proper orientation of the substrate in the active site, and furthermore this supports the proposed role of the stem peptide at binding subsite -2 in catalysis. Replacement of Gln100, a residue that would appear to interact with the N-acetyl group on MurNAc, did not show any changes in substrate affinity or activity.     

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.     

Purification and characterization of beta-1,6-glucanase of Streptomyces rochei application in the study of yeast cell wall proteins

A beta-1,6-glucanase was purified to apparent homogeneity from a commercial yeast digestive enzyme prepared from Streptomyces rochei by a series of column chromatographies. The molecular mass of the purified enzyme was 60 kDa by SDS-PAGE. The purified enzyme had an optimum pH range from 4.0 to 6.0 and was stable in the same pH range. The enzyme was stable under 50 degrees C but lost almost all activity at 60 degrees C. The enzyme was specific to beta-1,6-glucan and had little activity towards beta-1,3-glucan and beta-1,4-glucan. When the beta-1,6-glucan was hydrolyzed with the purified enzyme for 5 h, the reaction products contained 20% glucose, 36% gentiobiose, and 44% other oligosaccharides, suggesting that the enzyme is an endo-type glucanase. When the purified enzyme was used for the digestion of the cell wall of Saccharomyces cerevisiae, cell-wall proteins covalently bound to the cell-wall glucan were recovered as soluble forms, suggesting that this enzyme is useful for analysis of yeast-cell wall proteins.     

Characterization of a β-1,3-glucanase active in the alkaline midgut of Spodoptera frugiperda larvae and its relation to β-glucan-binding proteins

Spodoptera frugiperda β-1,3-glucanase (SLam) was purified from larval midgut. It has a molecular mass of 37.5 kDa, an alkaline optimum pH of 9.0, is active against β-1,3-glucan (laminarin), but cannot hydrolyze yeast β-1,3-1,6-glucan or other polysaccharides. The enzyme is an endoglucanase with low processivity (0.4), and is not inhibited by high concentrations of substrate. In contrast to other digestive β-1,3-glucanases from insects, SLam is unable to lyse Saccharomyces cerevisae cells. The cDNA encoding SLam was cloned and sequenced, showing that the protein belongs to glycosyl hydrolase family 16 as other insect glucanases and glucan-binding proteins. Multiple sequence alignment of β-1,3-glucanases and β-glucan-binding protein supports the assumption that the β-1,3-glucanase gene duplicated in the ancestor of mollusks and arthropods. One copy originated the derived β-1,3-glucanases by the loss of an extended N-terminal region and the β-glucan-binding proteins by the loss of the catalytic residues. SLam homology modeling suggests that E228 may affect the ionization of the catalytic residues, thus displacing the enzyme pH optimum. SLam antiserum reacts with a single protein in the insect midgut. Immunocytolocalization shows that the enzyme is present in secretory vesicles and glycocalyx from columnar cells.     

Enzyme-catalyzed formation of beta-peptides: beta-peptidyl aminopeptidases BapA and DmpA acting as beta-peptide-synthesizing enzymes

In recent studies, we discovered that the three beta-peptidyl aminopeptidases, BapA from Sphingosinicella xenopeptidilytica 3-2W4, BapA from S. microcystinivorans Y2, and DmpA from Ochrobactrum anthropi LMG7991, possess the unique feature of cleaving N-terminal beta-amino acid residues from beta- and alpha/beta-peptides. Herein, we investigated the use of the same three enzymes for the reverse reaction catalyzing the oligomerization of beta-amino acids and the synthesis of mixed peptides with N-terminal beta-amino acid residues. As substrates, we employed the beta-homoamino acid derivatives H-beta hGly-pNA, H-beta3 hAla-pNA, H-(R)-beta3 hAla-pNA, H-beta3 hPhe-pNA, H-(R)-beta3 hPhe-pNA, and H-beta3 hLeu-pNA. All three enzymes were capable of coupling the six beta-amino acids to oligomers with chain lengths of up to eight amino acid residues. With the enzyme DmpA as the catalyst, we observed very high conversion rates, which correspond to dimer yields of up to 76%. The beta-dipeptide H-beta3 hAla-beta3 hLeu-OH and the beta/alpha-dipeptide H-beta hGly-His-OH (carnosine) were formed with almost 50% conversion, when a five-fold excess of beta3-homoleucine or histidine was incubated with H-beta3 hAla-pNA and H-beta hGly-pNA, respectively, in the presence of the enzyme BapA from S. microcystinivorans Y2. BapA from S. xenopeptidilytica 3-2W4 turned out to be a versatile catalyst capable of coupling various beta-amino acid residues to the free N-termini of beta- and alpha-amino acids and even to an alpha-tripeptide. Thus, these aminopeptidases might be useful to introduce a beta-amino acid residue as an N-terminal protecting group into a 'natural' alpha-peptide, thereby stabilizing the peptide against degradation by other proteolytic enzymes.     

Molecular cloning and characterization of glucanase inhibitor proteins: coevolution of a counterdefense mechanism by plant pathogens

A characteristic plant response to microbial attack is the production of endo-beta-1,3-glucanases, which are thought to play an important role in plant defense, either directly, through the degradation of beta-1,3/1,6-glucans in the pathogen cell wall, or indirectly, by releasing oligosaccharide elicitors that induce additional plant defenses. We report the sequencing and characterization of a class of proteins, termed glucanase inhibitor proteins (GIPs), that are secreted by the oomycete Phytophthora sojae, a pathogen of soybean, and that specifically inhibit the endoglucanase activity of their plant host. GIPs are homologous with the trypsin class of Ser proteases but are proteolytically nonfunctional because one or more residues of the essential catalytic triad is absent. However, specific structural features are conserved that are characteristic of protein-protein interactions, suggesting a mechanism of action that has not been described previously in plant pathogen studies. We also report the identification of two soybean endoglucanases: EGaseA, which acts as a high-affinity ligand for GIP1; and EGaseB, with which GIP1 does not show any association. In vitro, GIP1 inhibits the EGaseA-mediated release of elicitor-active glucan oligosaccharides from P. sojae cell walls. Furthermore, GIPs and soybean endoglucanases interact in vivo during pathogenesis in soybean roots. GIPs represent a novel counterdefensive weapon used by plant pathogens to suppress a plant defense response and potentially function as important pathogenicity determinants.