A defence-related Olea europaea β-glucosidase hydrolyses and activates oleuropein into a potent protein cross-linking agent

Abstract

Oleuropein, the major secoiridoid compound in olive, is involved in a sophisticated two-component defence system comprising a β-glucosidase enzyme that activates oleuropein into a toxic glutaraldehyde-like structure. Although oleuropein deglycosylation studies have been monitored extensively, an oleuropein β-glucosidase gene has not been characterized as yet. Here, we report the isolation of OeGLU cDNA from olive encoding a β-glucosidase belonging to the defence-related group of terpenoid-specific glucosidases. In planta recombinant protein expression assays showed that OeGLU deglycosylated and activated oleuropein into a strong protein cross-linker. Homology and docking modelling predicted that OeGLU has a characteristic (β/α)8 TIM barrel conformation and a typical construction of a pocket-shaped substrate recognition domain composed of conserved amino acids supporting the β-glucosidase activity and non-conserved residues associated with aglycon specificity. Transcriptional analysis in various olive organs revealed that the gene was developmentally regulated, with its transcript levels coinciding well with the spatiotemporal patterns of oleuropein degradation and aglycon accumulation in drupes. OeGLU upregulation in young organs reflects its prominent role in oleuropein-mediated defence system. High gene expression during drupe maturation implies an additional role in olive secondary metabolism, through the degradation of oleuropein and reutilization of hydrolysis products.

The C-Domain of Oleuropein β-Glucosidase Assists in Protein Folding and Sequesters the Enzyme in Nucleus

“…Knowing that oleuropein aglycone exhibits strong protein-denaturing/protein-cross-linking properties (Koudounas et al, 2015), we asked whether the enzymatically produced oleuropein aglycone had any impact on the activity of the enzyme. Interestingly, no significant difference in the enzymatic activity could be observed between reactions incubated for 5, 10, or 15 min, in the presence or the absence of bovine serum albumin (BSA; i.e.…”

“…Accordingly, the OeGLU compartmentalization could allow the oleuropein/OeGLU dual-partner defense system to be inadequately active within olive cells where both OeGLU and oleuropein are highly accumulated (Amiot et al, 1986;Gutierrez-Rosales et al, 2012;Koudounas et al, 2015). In silico analysis of the OeGLU was predicted to contain a putative NLS at amino acid 542 to 550 (DRRKRLRGS) and no nuclear export signal (Supplemental Fig.…”

“…This product covalently binds to amino acids and exhibits strong protein denaturing/cross-linking activities ( Fig. 1; Konno et al, 1999;Uccella, 2000;Koudounas et al, 2015). The two components comprising a defensive glucoside and a specialized detonating b-glucosidase, usually separated in a distinct cellular compartment, are usually referred to as a "dual-partner defense system."…”

“…Nevertheless it is widely accepted that the enzymatic activity of b-glucosidase on oleuropein is the dominant reaction to shape the composition of virgin olive oil in secoiridoid derivatives, defining therefore the oil's nutritional and organoleptic properties (Obied et al, 2008;Romero-Segura et al, 2012;Hachicha Hbaieb et al, 2015). We have recently identified OeGLU, to our knowledge the first Glycoside Hydrolase Family 1 (GH1) b-glucosidase in Oleaceae able to activate oleuropein (Koudounas et al, 2015).…”

Deciphering the route to cyclic monoterpenes in Chrysomelina leaf beetles: source of new biocatalysts for industrial application?

“…To date, it has been shown that iridoid toxicity can be attributed to the highly reactive aglycones that are released from the corresponding nontoxic iridoid glucosides that are often safely stored in plant organelles [18,19]. Glycoside hydrolysis can be achieved nonenzymatically or enzymatically by β-glucosidases (hydrolases, EC 3.2.1.21) produced by the plants themselves [20][21][22] or by their enemies [12,13,23,24]. If the resulting compound is a reactive aldehyde, it has the ability to link irreversibly and nonselectively essential cellular components including proteins.…”