Structure-function relationships of maize b-glucosidase Zm.p60.1.

P. Mazura³, R. Dopitová², L. Janda², J. Damborský⁴, T. Filipi³,
N. S. Kiran³, B. Brzobohatý¹

¹Institute of Biophysics AS CR, v.v.i., CZ-61265, Brno, Czech Republic; ²Department of Functional Genomics and Proteomics, Masaryk University, CZ-62500 Brno, Czech Republic; ³Department of Molecular Biology and Radiobiology, Mendel University of Agriculture and Forestry, CZ-61300 Brno, Czech Republic; ⁴Loschmidt Laboratories, Masaryk University, CZ-62500 Brno, Czech Republic, mazura@sci.muni.cz

The basic structure of the vast majority of β-glucosidases is the (β/α)₈ barrel fold which is a fundamental structural element in many proteins. Proteins containing the (β/α)₈ barrel fold occur in five of the six general classes of catalytic activities according to the Enzyme Commission classification scheme.

Current knowledge as represented by more than 150 hits in the CaZy (carbohydrate active enzymes) database shows that the b-glucosidase (b-glucoside hydrolase, EC 3.2.1.21) class comprises extended “families” of enzymes hydrolyzing a broad variety of aryl- and alkyl- b -D-glucosides as well as glucosides made up only of carbohydrate moieties [1]. Widespread interest in b-glucosidase research reflects their essential functions in a variety of basic biological processes ranging from developmental regulation to chemical defense against pathogen attack, and in a number of industrial applications such as biomass conversion.

Our model enzyme, the maize b-glucosidase Zm-p60.1, is important for the regulation of plant development due to its role in the targeted release of free cytokinins from their inactive storage forms, cytokinin-O-glucosides [2,3].

Our research is focused on attempting to modulate enzyme specificity and understanding the functional relationships between key amino acid residues that form the entrance to the active site. We are interested in plant β-glucosidases and Zm-p60.1 is the best described among them. This group of proteins is highly diverse in terms of homologous enzymes, which enables bioinformatics as well as the elucidation of the biological significance of β-glucosidases.

In our work we have mainly used methods of rational design in protein engineering. that approach, however, has several limitations. Therefore we have adopted a strategy of random site directed mutagenesis followed by directed evolution to investigate the functional relationships between amino acid residues. Generally, this work will shed more light on the complex evolution of enzyme substrate interaction at the active site. Simultaneously, the ability to modulate specificity of β-glucosidases holds considerable promise in terms of biotechnological applications.

References:

[1] Berrin et al (2003) Substrate (aglycone) specificity of human cytosolic β-glucosidase Biochem. J. 373,41-48

[2] Brzobohatý et al (1993) Release of active cytokinin by a ß-glucosidase localized to the maize root meristem. Science. 262: 1051-1054.

[3] Kiran et al (2006) Ectopic over-expression of the maize b-glucosidase Zm-p60.1 perturbs cytokinin homeostasis in transgenic tobacco. J Exp Bot. 57(4): 985-96.

Acknowledgment:

This project was supported by grants GACR203/02/0865 from the Grant Agency of the Czech Republic, LC06034, LC06010, MSM0021622415 and MSM0021622412 from the Ministry of Education, Youth and Sports of the Czech Republic, and AVOZ50040507 from the Academy of Sciences of the Czech Republic.