Structural determinants investigation of endo and exo-arabinanases mode of action retrieved from bovine microflora

Author(s)

Carla Cristina Polo

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2016-01-27

Examining board members

Mário Tyago Murakami; Celso Eduardo Benedetti; Jörg Kobarg; Marcos Roberto Mattos Fontes; Hamilton Cabral

• Advisor: Mário Tyago Murakami

Abstract

The plant cell wall is composed by a polysaccharide complex together with structural proteins, which are able to hold and compress all the cytosolic organelles. Among these components, there are polymers such as cellulose, hemicellulose, pectin and lignin. To do so, the nature employs a wide range of specialized enzymes in the synthesis and degradation of these complex sugars, known as CAZymes (Carbohydrate-Active enZymes). Glycosyl hydrolases are among the most abundant CAZymes, represented by 135 families. The GH43 family is a polyspecific group of enzymes and contains a variety of ECs activities, for example that ones with arabinanase activity (EC 3.2.1.99), with application in different industrial fields. However, structural studies in order to elucidate the mechanisms underlying their regulation and mode of operation are still scarce. Their substrates, arabinans, are the second most abundant pentose in the plant cell wall, possessing considerable commercial value in food industry and biomass conversion. Therefore, the present study aimed the structural and biochemical characterization of two novel arabinanases, an endo-?-1,5-L-arabinanase (ARN2) and exo-?-1,5-L-arabinanase (ARN3) retrieved from the bovine rumen microflora and, which presented unique functional properties compared to their homologues. Their structures, solved by X-ray crystallography, revealed a canonical fold of the GH43 family, the 5-fold ?-propeller conserving the classical inversion mechanism that involves two conserved glutamic acid residues. Trough ARN2 characterization, it was possible to verify an extremely solvent accessible and large catalytic interface, explaining its preference to branched substrates and, which contrasts with the narrow interface of homologues that favors the binding of linear substrates. ARN3 also presented unusual features in its mode of action involving the recognition of the reducing end of the substrate and the release of arabinose. This region is found partially blocked by a long and divergent loop (R203-A230). To confirm the role of this region in the mode of action, a chimera was designed and produced and the long segment was substituted by the SRGEEP sequence that is conserved in classical GH43 endo-arabinanases. As predicted, the chimera acquired endo-enzyme properties, generating arabinobiose, mainly, as a final product. Interestingly, structural analyses also allowed to verify the role of the accessory domain in the stabilization of the catalytic cleft in two-domain enzymes such as ARN2. In single domain enzymes, such as ARN3, this region is stabilized by a pair of cysteines forming a disulfide bond. Furthermore, the ruminal enzymes showed a calcium-independent regulatory mechanism, an unheard behavior in the GH43 family. Finally, this work contributed collectively to new strategies to the hemicellulose/pectin degradation and expanded the current knowledge about GH43 family. In the post-text elements, there are the manuscript (I) related to the results presented in this thesis and the articles II and III from collaborative works. The final annex (IV) describes the work developed during the sandwich PhD at VTT Technical Research Centre of Finland (AU)

• FAPESP's process: 12/09119-8 - Structural determinants invertigation of endo and exo arabinanases mode of action from bovine rumen metagenomic
• Grantee: Carla Cristina Polo
• Support Opportunities: Scholarships in Brazil - Doctorate