Molecular Basis of Arabinobio-hydrolase Activity in Phytopathogenic Fungi

The phytopathogenic fungus Fusarium graminearum secretes a very diverse pool of glycoside hydrolases (GHs) aimed at degrading plant cell walls. α-l-Arabinanases are essential GHs participating in the complete hydrolysis of hemicellulose, a natural resource for various industrial processes, such as bioethanol or pharmaceuticals production. Arb93A, the exo-1,5-α-l-arabinanase of F. graminearum encoded by the gene fg03054.1, belongs to the GH93 family, for which no structural data exists. The enzyme is highly active (1065 units/mg) and displays a strict substrate specificity for linear α-1,5-l-arabinan. Biochemical assays and NMR experiments demonstrated that the enzyme releases α-1,5-l-arabinobiose from the nonreducing end of the polysaccharide. We determined the crystal structure of the native enzyme and its complex with α-1,5-l-arabinobiose, a degradation product of α-Me-1,5-l-arabinotetraose, at 1.85 and 2.05Å resolution, respectively. Arb93A is a monomeric enzyme, which presents the six-bladed β-propeller fold characteristic of sialidases of clan GHE. The configuration of the bound arabinobiose is consistent with the retaining mechanism proposed for the GH93 family. Catalytic residues were proposed from the structural analysis, and site-directed mutagenesis was used to validate their role. They are significantly different from those observed for GHE sialidases. The phytopathogenic fungus Fusarium graminearum secretes a very diverse pool of glycoside hydrolases (GHs) aimed at degrading plant cell walls. α-l-Arabinanases are essential GHs participating in the complete hydrolysis of hemicellulose, a natural resource for various industrial processes, such as bioethanol or pharmaceuticals production. Arb93A, the exo-1,5-α-l-arabinanase of F. graminearum encoded by the gene fg03054.1, belongs to the GH93 family, for which no structural data exists. The enzyme is highly active (1065 units/mg) and displays a strict substrate specificity for linear α-1,5-l-arabinan. Biochemical assays and NMR experiments demonstrated that the enzyme releases α-1,5-l-arabinobiose from the nonreducing end of the polysaccharide. We determined the crystal structure of the native enzyme and its complex with α-1,5-l-arabinobiose, a degradation product of α-Me-1,5-l-arabinotetraose, at 1.85 and 2.05Å resolution, respectively. Arb93A is a monomeric enzyme, which presents the six-bladed β-propeller fold characteristic of sialidases of clan GHE. The configuration of the bound arabinobiose is consistent with the retaining mechanism proposed for the GH93 family. Catalytic residues were proposed from the structural analysis, and site-directed mutagenesis was used to validate their role. They are significantly different from those observed for GHE sialidases. The plant cell wall consists mainly of a complex aggregation of polysaccharides, such as cellulose, hemicellulose, and pectin. Hemicellulose is one of the most abundant renewable biopolymers on earth and constitutes an important source of energy for the biofuel industry. It represents 20–40% of plant biomass and is principally composed of pentoses, such as xylose and arabinose (1Saha B.C. J. Ind. Microbiol. Biotechnol. 2003; 30: 279-291Crossref PubMed Scopus (1523) Google Scholar). Due to the high complexity and structural variability of this polysaccharide, many enzymes are necessary for its complete degradation (2Ward O.P. Moo-Young M. Crit. Rev. Biotechnol. 1989; 8: 237-274Crossref PubMed Scopus (101) Google Scholar). A number of microorganisms are able to break down hemicellulose, through the action of various glycoside hydrolases (GHs). 2The abbreviations used are: GH, glycoside hydrolase; PACE, polysaccharide analysis using carbohydrate gel electrophoresis; DP, degree of polymerization; WT, wild type. 2The abbreviations used are: GH, glycoside hydrolase; PACE, polysaccharide analysis using carbohydrate gel electrophoresis; DP, degree of polymerization; WT, wild type. The latter catalyze the cleavage of glycosidic bonds between sugars with either inversion or retention of the anomeric configuration (3Koshland D.E. Biol. Rev. 1953; 28: 416-436Crossref Scopus (803) Google Scholar). GHs have been classified into more than 114 different families based on their amino acid sequence similarity (CAZY (Carbohydrate Active Enzymes) server, available on the World Wide Web) (4Henrissat B. Biochem. Soc. Transact. 1998; 26: 153-156Crossref PubMed Scopus (151) Google Scholar, 5Cantarel B.L. Coutinho P.M. Rancurel C. Bernard T. Lombard V. Henrissat B. Nucleic Acids Res. 2009; 37: D233-D238Crossref PubMed Scopus (4067) Google Scholar). α-l-Arabinanases (EC 3.2.1.-) are accessory hemicellulases that hydrolyze α-l-arabinofuranosic linkages and act synergistically with other GHs to break down hemicellulose fully (6Margolles-Clark E. Tenkanen M. Nakari-Setala T. Penttila M. Appl. Environ. Microbiol. 1996; 62: 3840-3846Crossref PubMed Google Scholar). These enzymes have become of interest in recent years because of their potential rate-limiting role in the degradation of lignocelluloses and their practical application in various industrial processes, such as the production of important medicinal compounds, the improvement of wine flavors, pulp treatment, juice clarification, the production of bioethanol, and the synthesis of oligosaccharides (7Numan M.T. Bhosle N.B. J. Ind. Microbiol. Biotechnol. 2006; 33: 247-260Crossref PubMed Scopus (138) Google Scholar). According to the CAZY classification, α-l-arabinanases are present in six GH families (3, 43, 51, 54, 62, and 93) whose members display highly varying specificities. GH51 and GH54 are the most extensively characterized and contain mainly α-l-arabinofuranosidases, removing both α-1,2 and α-1,3 arabinofuranosyl moieties from arabinan and xylans (8Hovel K. Shallom D. Niefind K. Belakhov V. Shoham G. Baasov T. Shoham Y. Schomburg D. EMBO J. 2003; 22: 4922-4932Crossref PubMed Scopus (119) Google Scholar, 9Miyanaga A. Koseki T. Matsuzawa H. Wakagi T. Shoun H. Fushinobu S. J. Biol. Chem. 2004; 279: 44907-44914Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 10Paes G. Skov L.K. O'Donohue M.J. Remond C. Kastrup J.S. Gajhede M. Mirza O. Biochemistry. 2008; 47: 7441-7451Crossref PubMed Scopus (45) Google Scholar, 11Taylor E.J. Smith N.L. Turkenburg J.P. D'Souza S. Gilbert H.J. Davies G.J. Biochem. J. 2006; 395: 31-37Crossref PubMed Scopus (77) Google Scholar). GH3 arabinofuranosidases are all bifunctional proteins acting on both β-1,4-xylose-xylose and β-1,3-xylose-arabinose bonds (12Mai V. Wiegel J. Lorenz W.W. Gene (Amst.). 2000; 247: 137-143Crossref PubMed Scopus (58) Google Scholar). GH62 enzymes have a specific arabinofuranose-debranching activity on xylan (13Gielkens M.M. Visser J. de Graaff L.H. Curr. Genet. 1997; 31: 22-29Crossref PubMed Scopus (67) Google Scholar), whereas GH43 and GH93 proteins are known to hydrolyze α-1,5-linked arabinofuranose-oligosaccharides (14Nurizzo D. Turkenburg J.P. Charnock S.J. Roberts S.M. Dodson E.J. McKie V.A. Taylor E.J. Gilbert H.J. Davies G.J. Nat. Struct. Biol. 2002; 9: 665-668Crossref PubMed Scopus (154) Google Scholar, 15Sakamoto T. Thibault J.F. Appl. Environ. Microbiol. 2001; 67: 3319-3321Crossref PubMed Scopus (40) Google Scholar). Nine three-dimensional structures ofα-l-arabinanases belonging to the GH43, -51, and -54 families have been solved to date (8Hovel K. Shallom D. Niefind K. Belakhov V. Shoham G. Baasov T. Shoham Y. Schomburg D. EMBO J. 2003; 22: 4922-4932Crossref PubMed Scopus (119) Google Scholar, 9Miyanaga A. Koseki T. Matsuzawa H. Wakagi T. Shoun H. Fushinobu S. J. Biol. Chem. 2004; 279: 44907-44914Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 10Paes G. Skov L.K. O'Donohue M.J. Remond C. Kastrup J.S. Gajhede M. Mirza O. Biochemistry. 2008; 47: 7441-7451Crossref PubMed Scopus (45) Google Scholar, 11Taylor E.J. Smith N.L. Turkenburg J.P. D'Souza S. Gilbert H.J. Davies G.J. Biochem. J. 2006; 395: 31-37Crossref PubMed Scopus (77) Google Scholar, 14Nurizzo D. Turkenburg J.P. Charnock S.J. Roberts S.M. Dodson E.J. McKie V.A. Taylor E.J. Gilbert H.J. Davies G.J. Nat. Struct. Biol. 2002; 9: 665-668Crossref PubMed Scopus (154) Google Scholar, 16Brunzelle J.S. Jordan D.B. McCaslin D.R. Olczak A. Wawrzak Z. Arch. Biochem. Biophys. 2008; 474: 157-166Crossref PubMed Scopus (45) Google Scholar, 17Proctor M.R. Taylor E.J. Nurizzo D. Turkenburg J.P. Lloyd R.M. Vardakou M. Davies G.J. Gilbert H.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2697-2702Crossref PubMed Scopus (78) Google Scholar, 18Vandermarliere E. Bourgois T.M. Winn M.D. Van Campenhout S. Volckaert G. Delcour J.A. Strelkov S.V. Rabijns A. Courtin C.M. Biochem. J. 2009; 418: 39-47Crossref PubMed Scopus (68) Google Scholar, 19Yamaguchi A. Tada T. Wada K. Nakaniwa T. Kitatani T. Sogabe Y. Takao M. Sakai T. Nishimura K. J. Biochem. (Tokyo). 2005; 137: 587-592Crossref PubMed Scopus (31) Google Scholar). Barley glucanases, which are members of GH3, have been structurally characterized (20Varghese J.N. Hrmova M. Fincher G.B. Full Text Full Text PDF PubMed Scopus Google Scholar), and those of GH62 are to display the β-propeller fold as the GH43 (14Nurizzo D. Turkenburg J.P. Charnock S.J. Roberts S.M. Dodson E.J. McKie V.A. Taylor E.J. Gilbert H.J. Davies G.J. Nat. Struct. Biol. 2002; 9: 665-668Crossref PubMed Scopus (154) Google Scholar). GH93 is as a of in the CAZY data from and and the characterized enzyme is from arabinobiose from the nonreducing end of linear arabinan T. Thibault J.F. Appl. Environ. Microbiol. 2001; 67: 3319-3321Crossref PubMed Scopus (40) Google Scholar, T. H. A. H. H. 2004; PubMed Scopus Google Scholar). The of its activity retention of the configuration of the anomeric T. T. H. Biophys. 2004; PubMed Scopus Google Scholar). GH93 is the of α-l-arabinanases for which structural been to Fusarium graminearum is a fungus that various such as or the the fungus secretes a and diverse of GHs that the cell F. graminearum in with plant cell wall as the as many as CAZY families and are the enzymes V. F. C. F. D. E. Curr. Genet. 2005; PubMed Scopus Google Scholar). The is high for enzymes hemicellulose, as by a analysis that are in to plant cell wall D. V. E. Biochem. Biophys. Res. 2006; PubMed Scopus Google Scholar). of the F. graminearum data U. G. M. D. M. V. G. Nucleic Acids Res. 2006; PubMed Scopus Google and are to an exo-1,5-α-l-arabinanase of structure with enzymes of clan which families and clan is characterized by a six-bladed β-propeller fold and (EC or (EC or catalyze the of acid residues from various and are in of many and T. PubMed Scopus Google Scholar). the and structural analysis of Arb93A, the exo-1,5-α-l-arabinanase from F. graminearum encoded by the gene We the structure of the native enzyme and its complex with α-1,5-l-arabinobiose, which to are the crystal structures of a GH93 data that the enzyme linear and releases α-1,5-l-arabinobiose from the nonreducing end with retention of the anomeric The residues through of the active were by site-directed We a mechanism for hydrolysis and Arb93A structure with that of enzymes of the clan GHE. and was from and with of from to were from F. graminearum was from and by F. graminearum was at on D.B. K. 1997; PubMed Scopus Google with cell wall as the source at a of The was as V. F. C. F. D. E. Curr. Genet. 2005; PubMed Scopus Google Scholar). and were used for site-directed and respectively. E. were in with and of was from F. graminearum with the to the was from the of the using the synthesis and the The were used for of with the and for The was using The product was on a and into the with and using The the gene in with the sequence of an of Arb93A were using a site-directed mutagenesis to the and using as The were used to the and for and for and for The were by of Arb93A and with the wild or gene were in of and was with at an of was for at A was to the of S. 1997; PubMed Scopus Google Scholar). were by and in and The was at for and a with and in the with of and the was with of the with a with the was a with The the of interest was a in the a with of and the was with of the with The was at with at in and in a of The was with through a to and a with and with of and the was with and in highly that were used for The production was The was by The was the that all were with was with and were using Arb93A and substrate in at the specificity of the enzyme, various were the of was determined by at using as the Hrmova M. J. Fincher G.B. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). and the of sugars was determined with the acid Chem. 31: Scopus Google Scholar), using α-1,5-l-arabinobiose as a of enzyme activity was as the of enzyme of α-1,5-l-arabinobiose the of and the were with as from to and from to The enzyme was with the substrate in for and for and and for of and used in NMR assays and were as in the to those used for the of other M. Y. J. Chem. Soc. PubMed Scopus Google Scholar, H. D'Souza J. Chem. 2002; 67: PubMed Scopus Google Scholar). were with and with from to as were enzyme in a of of with substrate from to at the were by to were used for with acid and analysis by polysaccharide analysis using carbohydrate gel as C. V. 2009; PubMed Scopus Google Scholar). the of were with the were in were using of the NMR and were by at from into was for the enzyme by and on a NMR were with a at a of in NMR were with data and to A substrate was the enzyme, and experiments were of and and of to in and were to of to a of for the enzyme and for the A was and for and a was at the of enzyme to in were to of in the to a of for the enzyme and for the A was and for and for at was using the as and an of was used for one and were by the using a of with at were with a and and the at were with a and the at The complex was in and for at with was as necessary to in at K. and complex data were on a crystal at the on and using were using J. Appl. 26: Scopus Google and to structure using in the Biol. PubMed Scopus Google Scholar). was using the for data are in and in to the bonds in to the in a and a for Arb93A were by the using the and C. E. G. Biol. 2006; Scholar). were and with a of with an of in and in which a A. Nat. Struct. Biol. PubMed Scopus Google Scholar). was used to the structure of the native by using J. 1997; PubMed Scopus Google Scholar). The was with The complex structure was solved by using and the native as a Biol. 2005; PubMed Scopus Google Scholar). The structures were using Dodson E.J. Biol. 1997; PubMed Scopus Google with in K. Biol. 2004; PubMed Scopus Google Scholar). The of the was of the were using and The of the was with the J. Appl. 26: Google to in the the and for the native and complex respectively. of the are in were with the and sequence from of F. graminearum was in and to amino The was to an with a cleavage between and the the of amino was in for production in E. The was consistent with the of A in the that the structurally characterized to Arb93A was the from with sequence A. S. Taylor G. Full Text Full Text PDF PubMed Scopus Google Scholar). and analysis at different and an activity at and the of The is highly in a of and more than of activity is at and the substrate the activity of Arb93A was on different The is active on that more than one is necessary for was by analysis of the hydrolysis from with that the of the sugars was Arb93A is able to arabinose or of a xylan because no activity was on of sugars was observed with arabinan and of and respectively. to the α-1,5-linked of is by The in activity by the that are by arabinobiose and arabinose are that the enzyme the substrate in an of Arb93A as an exo-1,5-α-l-arabinanase α-1,5-l-arabinobiose a product of to its from T. T. H. Biophys. 2004; PubMed Scopus Google specificity of Arb93A at and of enzyme activity was as the of enzyme of α-1,5-l-arabinobiose in a of Arb93A with and arabinan were at and The are very for all of the varying between and the observed of are to the in for the of the enzyme for the to with the of the for which the enzyme displays a of is more to hydrolysis than the other is no between the of Arb93A for and of the of the native from with that of Arb93A that Arb93A activity is than activity on T. J. Appl. 2006; Google of Arb93A on with various and to α-1,5-l-arabinobiose, and from were at and in a NMR from the cleavage of by Arb93A was in by a to arabinobiose S. A. S. Scopus Google The at a very that to to this It was to the retention or inversion mechanism of the enzyme by this the to the a whereas a with the of at the end of the arabinobiose and were present in the demonstrated that the hydrolysis by the nonreducing end and that an from A with substrate was at and to down the enzyme arabinobiose and were the of an arabinobiose for cleavage of the this substrate and was to the configuration of the anomeric glycosidic to down the and the by the at the and an essential of the Arb93A crystal structure was solved by the using a because with different The native structure was determined and fully to 1.85 the crystal as in Arb93A is a monomeric of a of amino is observed in the and all from and in the The presents a six-bladed β-propeller fold is of a The in the of the and the β-propeller by the observed for this fold Z. M. 2002; Full Text Full Text PDF PubMed Scopus Google The and by residues of the The a A acid at the is in the active of a from sequence the for structural of Arb93A similarity with the from clan GHE. The as determined by E. K. Biol. 2004; PubMed Scopus Google Scholar), are sialidases of the the one from M. and the enzyme of and for and A. S. Taylor G. Full Text Full Text PDF PubMed Scopus Google Scholar, C. B. G. E. S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Arb93A presents to the of sialidases in the and B. J. J. 1989; PubMed Scopus Google Scholar). The role of this in sialidases and in structurally proteins Sci. 2001; PubMed Scopus Google Scholar). They are from the active and have been proposed to the of the β-propeller A. S. Taylor G. Full Text Full Text PDF PubMed Scopus Google Scholar). of Arb93A crystal with to The complex structure solved by the of an α-1,5-l-arabinobiose in the of the of the residues from the the residues of the have been from the active and are of is observed for the active between and and and and in to the The between the native and complex structure is The active is in a at the of the β-propeller are by and were as and moieties an that to a energy for this S. S. A. Res. Scopus Google Scholar). The active a that to the It in a more is to The is and is by residues and and residues and The is with no of the at the nonreducing the no which the Arb93A for linear the the bonds with and The with whereas of the and are by with the for the and the and the for the the the glycosidic presents an to the The is to the the is to and the is to and and the anomeric is to have been with in the and in the as as a with at the of the glycosidic The anomeric of the a The hydrolysis from the nonreducing end is by structural The is consistent with a which the retention of the configuration of the anomeric proposed for its T. T. H. Biophys. 2004; PubMed Scopus Google of between arabinobiose and the in a Catalytic and essential residues are in the retention mechanism by (3Koshland D.E. Biol. Rev. 1953; 28: 416-436Crossref Scopus (803) Google Scholar), an and a of the amino to the anomeric that and act as the residues of is the glycosidic in a for The of is in with a role and is to the anomeric The residues are which is in the observed for other retaining enzymes Curr. Struct. Biol. PubMed Scopus Google Scholar). of the clan GHE a as the and structure between Arb93A and sialidases that an is present in the Arb93A active to the is in a for It is the anomeric on the and with the It a role in that of the residues was to and of the were with the linear as the and no displays a activity of with that of displays analysis of the of by Arb93A and its is by of the no hydrolysis of arabinofuranose-oligosaccharides were observed with and was active on arabinofuranose-oligosaccharides with of and whereas was able to with of and Arb93A with the other GH93 from F. graminearum and from T. H. A. H. H. 2004; PubMed Scopus Google and from Fusarium that the residues are in those enzymes a role of and in the whereas the role of the We a retaining mechanism for with as an and as a as in According to the CAZY classification, GHs are in 114 families based on their sequence on the World Wide of no three-dimensional structure been solved to structures of native GHs and their complex with the and We the structure of a GH93 an from the fungus F. with to its six-bladed β-propeller Arb93A into clan GHE. this clan of the which with most and sialidases and and families and which contain sialidases and respectively. The of the Arb93A structure with its the from M. A. S. Taylor G. Full Text Full Text PDF PubMed Scopus Google Scholar), is in and to other sialidases. from the six-bladed β-propeller Arb93A presents with M. of is that Arb93A a carbohydrate carbohydrate have been proposed to a role in the of the S. B. A. G.J. J. Chem. Soc. 2003; PubMed Scopus Google Scholar), the of the using as substrate is of the of as the of Arb93A on the different J.N. Biochemistry. 2006; PubMed Scopus Google Scholar). both of the active is in an at the of the The Arb93A active is more a whereas sialidases one The active is more in The of the active of sialidases that with the of acid is highly which is observed in Arb93A as to the of a in its Active and and structural data that Arb93A the nonreducing arabinobiose from linear the complex crystal the nonreducing of the is and because the at is the most a polysaccharide substrate from the end glycosidic of the with an in the of the The active is more the at the is no for a is a carbohydrate The present that of the GH93 act with retention of a mechanism that is observed in sialidases. the for retention of configuration was observed in the of its activity T. T. H. Biophys. 2004; PubMed Scopus Google Scholar). and in to the enzymes of present a that is different from the sialidases Davies G.J. Curr. Chem. Biol. 2008; PubMed Scopus Google Scholar). and data that Arb93A through a retaining mechanism and acting as and and The enzyme classified as a such as those of the clan Chem. PubMed Google Scholar). on the other a as the in Davies G.J. Curr. Chem. Biol. 2008; PubMed Scopus Google Scholar). It is the the that are in sialidases that is present in members of the in Arb93A, for to a of the different of the active which is more in The of sialidases is at the of the of Arb93A The anomeric in Arb93A is more than from the observed in sialidases. on the of the anomeric and as for a The in the of the residues and have in to the of the different A. T. A. G. P.M. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). of of to in a of activity of which that this is important for activity role is that this the by the as observed for other enzymes of the clan such as from or of E. G. M. Z. M. C. Davies G.J. Chem. Biol. Full Text PDF PubMed Scopus Google Scholar, A. M. Davies G.J. J. Biol. 2000; PubMed Scopus Google Scholar). It to the of the in the the structure is The from the in the present of the this have a role in the of the substrate in the The is active on the and hydrolyze a the active a between the and by and The with the in the and with in the the the the whereas for in for the of in the from an the substrate in the active and its to to a activity (14Nurizzo D. Turkenburg J.P. Charnock S.J. Roberts S.M. Dodson E.J. McKie V.A. Taylor E.J. Gilbert H.J. Davies G.J. Nat. Struct. Biol. 2002; 9: 665-668Crossref PubMed Scopus (154) Google Scholar). with been demonstrated that F. graminearum GH93 α-l-arabinanases sequence and V. The for the of enzymes to catalyze the is These proteins display substrate a of the fungus to its Arb93A displays strict substrate specificity for with of and α-1,5-l-arabinobiose from the The of the arabinan and used for activity is through to of a The observed hydrolysis through the nonreducing end of the is with hydrolysis of such were observed for of sequence with Arb93A T. Thibault J.F. Appl. Environ. Microbiol. 2001; 67: 3319-3321Crossref PubMed Scopus (40) Google Scholar, T. T. H. Biophys. 2004; PubMed Scopus Google Scholar). from the GH93 present the and their of action specific to this with the of the in the of the mechanism for the GH93 experiments with to the retention to the both at the enzyme and substrate and the of The of the structural and of the GH93 enzymes as as the of their is of at the industrial It in the of the degradation of the highly diverse present in Arb93A represents a the of a of which is of interest for a of renewable natural We are very to for the We the for to data and for NMR with

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