Benedita A Pinheiro

Publications (5)20.14 Total impact

• Article: Putting an N-terminal end to the Clostridium thermocellum xylanase Xyn10B story: crystal structure of the CBM22-1-GH10 modules complexed with xylohexaose.

  Shabir Najmudin, Benedita A Pinheiro, José A M Prates, Harry J Gilbert, Maria J Romão, Carlos M G A Fontes
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  ABSTRACT: In general, plant cell wall degrading enzymes are modular proteins containing catalytic domains linked to one or more non-catalytic carbohydrate-binding modules (CBMs). Xyn10B from Clostridium thermocellum is a typical modular enzyme containing an N-terminal family 22 CBM (CBM22-1), a family 10 glycoside hydrolase catalytic domain (GH10), a second CBM22 (CBM22-2), a dockerin sequence and a C-terminal family 1 carbohydrate esterase (CE1) catalytic domain. The structure of the N-terminal bi-modular CBM22-1-GH10 component of Xyn10B has been determined using a SeMet derivative by SAD to 2.5Å. The data was extended to 2.0Å for the non-SeMet mutant complexed with xylohexaose. CBM22-1-GH10 is a 60kDa protein with an E337A mutation to render the GH10 subunit inactive. Three of the six xylose residues of xylohexaose are shown to be bound in the inactivated GH10 substrate binding cleft, with the other three sugars presumably disordered in the solvent channel. The protein is a dimer in the asymmetric unit with extensive surface contacts between the two GH10 modules and between the CBM22-1 and GH10 modules. Residues from helix H4 of the GH10 module provide the major contacts by fitting into the minor groove of the CBM22-1 module. The orientation of CBM22-1 is such that it would allow the substrate to be loosely bound and subsequently delivered to the active site in a processive manner.
  Journal of Structural Biology 12/2010; 172(3):353-62. · 3.41 Impact Factor
• Source

  Available from: José A M Prates

  Article: The active site of a carbohydrate esterase displays divergent catalytic and noncatalytic binding functions.

  Cedric Montanier, Victoria A Money, Virginia M R Pires, James E Flint, Benedita A Pinheiro, Arun Goyal, José A M Prates, Atsushi Izumi, Henrik Stålbrand, Carl Morland, Alan Cartmell, Katarina Kolenova, Evangelos Topakas, Eleanor J Dodson, David N Bolam, Gideon J Davies, Carlos M G A Fontes, Harry J Gilbert
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  ABSTRACT: Multifunctional proteins, which play a critical role in many biological processes, have typically evolved through the recruitment of different domains that have the required functional diversity. Thus the different activities displayed by these proteins are mediated by spatially distinct domains, consistent with the specific chemical requirements of each activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within an enzyme is likely to be a rare event, because it would compromise the existing activity of the protein. In contrast to this view, a potential example of multifunctional recruitment into a single protein domain is provided by CtCel5C-CE2, which contains an N-terminal module that displays cellulase activity and a C-terminal module, CtCE2, which exhibits a noncatalytic cellulose-binding function but also shares sequence identity with the CE2 family of esterases. Here we show that, unlike other CE2 members, the CtCE2 domain displays divergent catalytic esterase and noncatalytic carbohydrate binding functions. Intriguingly, these diverse activities are housed within the same site on the protein. Thus, a critical component of the active site of CtCE2, the catalytic Ser-His dyad, in harness with inserted aromatic residues, confers noncatalytic binding to cellulose whilst the active site of the domain retains its esterase activity. CtCE2 catalyses deacetylation of noncellulosic plant structural polysaccharides to deprotect these substrates for attack by other enzymes. Yet it also acts as a cellulose-binding domain, which promotes the activity of the appended cellulase on recalcitrant substrates. The CE2 family encapsulates the requirement for multiple activities by biocatalysts that attack challenging macromolecular substrates, including the grafting of a second, powerful and discrete noncatalytic binding functionality into the active site of an enzyme. This article provides a rare example of "gene sharing," where the introduction of a second functionality into the active site of an enzyme does not compromise the original activity of the biocatalyst.
  PLoS Biology 04/2009; 7(3):e71. · 11.45 Impact Factor
• Article: Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-beta-D-xylanase 10B in complex with xylohexaose.

  Shabir Najmudin, Benedita A Pinheiro, Maria J Romão, José A M Prates, Carlos M G A Fontes
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  ABSTRACT: The cellulosome of Clostridium thermocellum is a highly organized multi-enzyme complex of cellulases and hemicellulases involved in the hydrolysis of plant cell-wall polysaccharides. The bifunctional multi-modular xylanase Xyn10B is one of the hemicellulase components of the C. thermocellum cellulosome. The enzyme contains an internal glycoside hydrolase family 10 catalytic domain (GH10) and a C-terminal family 1 carbohydrate esterase domain (CE1). The N-terminal moiety of Xyn10B (residues 32-551), comprising a carbohydrate-binding module (CBM22-1) and the GH10 E337A mutant, was crystallized in complex with xylohexaose. The crystals belong to the trigonal space group P3(2)21 and contain a dimer in the asymmetric unit. The crystals diffracted to beyond 2.0 A resolution.
  Acta Crystallographica Section F Structural Biology and Crystallization Communications 09/2008; 64(Pt 8):715-8. · 0.51 Impact Factor
• Article: The Clostridium cellulolyticum dockerin displays a dual binding mode for its cohesin partner.

  Benedita A Pinheiro, Mark R Proctor, Carlos Martinez-Fleites, José A M Prates, Victoria A Money, Gideon J Davies, Edward A Bayer, Carlos M G A Fontesm, Henri-Pierre Fierobe, Harry J Gilbert
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  ABSTRACT: The plant cell wall degrading apparatus of anaerobic bacteria includes a large multienzyme complex termed the "cellulosome." The complex assembles through the interaction of enzyme-derived dockerin modules with the multiple cohesin modules of the noncatalytic scaffolding protein. Here we report the crystal structure of the Clostridium cellulolyticum cohesin-dockerin complex in two distinct orientations. The data show that the dockerin displays structural symmetry reflected by the presence of two essentially identical cohesin binding surfaces. In one binding mode, visualized through the A16S/L17T dockerin mutant, the C-terminal helix makes extensive interactions with its cohesin partner. In the other binding mode observed through the A47S/F48T dockerin variant, the dockerin is reoriented by 180 degrees and interacts with the cohesin primarily through the N-terminal helix. Apolar interactions dominate cohesin-dockerin recognition that is centered around a hydrophobic pocket on the surface of the cohesin, formed by Leu-87 and Leu-89, which is occupied, in the two binding modes, by the dockerin residues Phe-19 and Leu-50, respectively. Despite the structural similarity between the C. cellulolyticum and Clostridium thermocellum cohesins and dockerins, there is no cross-specificity between the protein partners from the two organisms. The crystal structure of the C. cellulolyticum complex shows that organism-specific recognition between the protomers is dictated by apolar interactions primarily between only two residues, Leu-17 in the dockerin and the cohesin amino acid Ala-129. The biological significance of the plasticity in dockerin-cohesin recognition, observed here in C. cellulolyticum and reported previously in C. thermocellum, is discussed.
  Journal of Biological Chemistry 07/2008; 283(26):18422-30. · 4.77 Impact Factor
• Article: Putting an N-terminal end to the Clostridium thermocellum xylanase Xyn10B story: Crystal structure of the CBM22-1–GH10 modules complexed with xylohexaose

  Shabir Najmudin, Benedita A. Pinheiro, José A.M. Prates, Harry J. Gilbert, Maria J. Romão, Carlos M.G.A. Fontes
  [show abstract] [hide abstract]
  ABSTRACT: In general, plant cell wall degrading enzymes are modular proteins containing catalytic domains linked to one or more non-catalytic carbohydrate-binding modules (CBMs). Xyn10B from Clostridium thermocellum is a typical modular enzyme containing an N-terminal family 22 CBM (CBM22-1), a family 10 glycoside hydrolase catalytic domain (GH10), a second CBM22 (CBM22-2), a dockerin sequence and a C-terminal family 1 carbohydrate esterase (CE1) catalytic domain. The structure of the N-terminal bi-modular CBM22-1–GH10 component of Xyn10B has been determined using a SeMet derivative by SAD to 2.5 Å. The data was extended to 2.0 Å for the non-SeMet mutant complexed with xylohexaose. CBM22-1–GH10 is a 60 kDa protein with an E337A mutation to render the GH10 subunit inactive. Three of the six xylose residues of xylohexaose are shown to be bound in the inactivated GH10 substrate binding cleft, with the other three sugars presumably disordered in the solvent channel. The protein is a dimer in the asymmetric unit with extensive surface contacts between the two GH10 modules and between the CBM22-1 and GH10 modules. Residues from helix H4 of the GH10 module provide the major contacts by fitting into the minor groove of the CBM22-1 module. The orientation of CBM22-1 is such that it would allow the substrate to be loosely bound and subsequently delivered to the active site in a processive manner.
  Journal of Structural Biology.