Purification and characterization of digestive beta-glycosidases from Spodoptera frugiperda (Lepidoptera)

Author(s)

Sandro Roberto Marana

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 1999-04-05

Examining board members

Clélia Ferreira Terra; Pedro Soares de Araújo; Ângelo Geraldo Gambarini; Osvaldo Marinotti; Helena Bonciani Nader

• Advisor: Clelia Ferreira Terra

Abstract

Two digestive &#946;-glycosidases (MW 47,000 and 50,000, named &#946;gly47 and &#946;gly50, respectively) whose are found in the S. frugiperda larvae were purified by a combination of chromatographic steps. Substrate competition experiments and chemical modification data showed that &#946;gly47 has two active sites. One of them was called aryl &#946;-glycosidase and presents a -1 subsite that prefers galactose while the +1 subsite binds small cyclic hydrophobic groups. The other active site was called cellobiase and presents 4 subsites that bind glucose residues weaker as they get far from the cleavage point. The cDNA that codes the &#946;gly50 was cloned and sequenced. Amino acid sequence alignment, substrate competition experiments and inhibitions proved that this enzyme has just one active site. The -1 subsite specificity is controlled by a hydrogen bond network as it was showed comparing the kinetic parameters (Kcat and KcatlKm) for some NP&#946;glycosides hydrolysis. The aglycone binding region, a hydrophobic cleft, was studied with alkyl &#946;-glucosides and oligocellodextrins as competitive inhibitors. Amino acid sequence alignment between the &#946;gly50 and other glycosil hydrolases showed the amino acids responsible for the substrate binding and that the GIU<SUB.187 (proton donor - pKa = 7.5) and GIU399 (nucleophile - pKa = 4.5) are directly involved in the catalysis. Beside this, Arg97 and Tyr331 participate indirectly in the catalysis, modulating the nucleophile pKa (AU)