New Uses for Glucuronidase Genes and Proteins

Historically CAMBIA is strongly associated with the ß-glucuronidase gene (gusA) from E.coli. There are thousands of publications documenting its extensive use and versatility as a marker gene for plant genetic transformation and molecular physiology studies.

More recently the hydrolytic capabilities of the gusA gene have been exploited to release aglycones from glucuronides. This principle can be exploited widely either to increase phloem transportability of hydrophobic substances and/or to reactivate inert, biochemical activity compounds to development of novel, second-generation hydrolytic enzymes with improved characteristics (e.g. secretability into the apoplast of plant tissues, improved higher thermal and chemical stability to expand on existing histochemical assay conditions, variants in substrate specificity and processability).

A different approach consists in the development of transport mechanisms based on substrate-specific permeases, to trap the substrates in the cell. Phloem-translocatable, bioactive pro-compounds are being developed as target substrates for the activating enzymes.

Primer on Glucuronide Metabolism

Glucuronidation is one of the main detoxification pathways of xenobiotics but also self-metabolites in vertebrates. Microbial intestinal flora and other microorganisms have developed hydrolytic capabilities to obtain the glucuronate moiety as a carbon source.

In E. coli, an operon constituted of four genes, encoding a repressor, a glucuronidase, a permease, and a porin-like protein, is responsible for import and hydrolysis of glucuronide. Free glucuronate is then funnelled into metabolic pathways.

Substrates

The development of novel substrates for tissue-specific hydrolases, substrates having specific chemical, biochemical and physiological aspects, can contribute to selection and synthesis of agrichemical glucuronides, development of derivatives for positive selection, gametocidic or toxic chemicals.

Candidates have been selected based on their chemical suitability to provide derivatives. Another criterion is inactivity as a glucuronide and also non-toxicity to humans and non-target organisms. This also reduces the exposure of non-target pests to pesticides, reducing resistance buildup.

Another advantage of the approach is that the attachment of glucuronic acid makes many substances phloem translocatable, widening the scope of applicability for many agrichemicals whose use, although ecologically compatible, has been restricted by the lack of phloem mobility.