The role of cysteine residues in structure and enzyme activity of a maize β-glucosidase

Abstract

The maize Zm-p60.1 gene encodes a β-glucosidase that can release active cytokinins from their storage forms, cytokinin-O-glucosides. Mature catalytically active Zm-p60.1 is a homodimer containing five cysteine residues per a subunit. Their role was studied by mutating them to alanine (A), serine (S), arginine (R) or aspartic acid (D) using site-directed mutagenesis, and subsequent heterologous expression in Escherichia coli. All substitutions of C205 and C211 resulted in decreased formation and/or stability of the homodimer, manifested as accumulation of high levels of monomer in the bacterial expression system. Examination of urea- and glutathione-induced dissociation patterns of the homodimer to the monomers, HPLC profiles of hydrolyric fragments of reduced and oxidized forms, and a homology-based three-dimensional structural model revealed that an intramolecular disulfide bridge formed between C205 and C211 within the subunits stabilized the quaternary structure of the enzyme. Mutating C52 to R produced a monomeric enzyme protein, too. No detectable effects on homodimer formation were apparent in C170 and C479 mutants. Given the K(m) values for C170A/S mutants were equal to that for the wild-type enzyme, C170 cannot participate in enzyme-substrate interactions. Possible indirect effects of C170A/S mutations on catalytic activity of the enzyme were inferred from slight decreases in the apparent catalytic activity, k'(cat). C170 is located on a hydrophobic side of an α-helix packed against hydrophobic aminoacid residues of β-strand 4, indicating participation of C170 in stabilization of a (β/α)8 barrel structure in the enzyme. In C479A/D/R/S mutants, K(m) and k'(cat) were influenced more significantly suggesting a role for C479 in enzyme catalytic action.