Abstract:
The horticulture industry loses millions in post-harvest waste from the spoilage of over-ripe produce every year. Ripening and spoilage are, in large part, due to pectinase enzymes such as polygalacturonase (PG)
and pectin lyase-likes (PLLs) which are responsible for different modes of pectin degradation within the plant cell wall (PCW). Whilst plant pectinases help regulate and maintain growth and developmental processes,
fungal and bacterial pectinases are involved in pathogenic diseases such as Verticillium wilt. Pectinases have been widely used in industrial applications, but their mechanisms of specificity are largely unknown. Such knowledge would not only allow fine control of pectinases in industry, but also help combat plant parasitism. Identification of an abundance of pectinase isoforms within Arabidopsis thaliana raised questions regarding
isoform redundancy. This study aimed to utilise molecular dynamics simulations coupled with biochemical assays, to characterise the different enzyme-substrate behaviours of two PG isoforms – ADPG2 and PGLR
– to address this supposed redundancy, with comparisons to a PLL from Verticillium dahliae that exhibits dual activity on two different substrate types.
Analysis of enzyme activity, complex dynamics, and protein-substrate interactions within various complexes, found that subtle differences between substrate binding profiles determined enzyme specificity on differently
methylesterified substrates. Unique disulphide bond forming cysteine residues, located within disordered loop regions flanking the enzyme binding grooves, were found to negatively impact substrate dynamics under
reducing conditions. Results concluded that enzyme processivity, and thus activity, is reliant on the motional
freedom of bound substrates – influenced by binding groove interactions, protein dynamics and likely redox
homeostasis of the PCW. Analysis of Ca2+ dependent binding of VdPelB to differently methylesterified
substrates, attributed the dual action to the pathogenic nature of V. dahliae. Hypothesised PG redundancy was rejected upon characterisation of individual binding differences that illustrate distinctive pectin fine tuning behaviour unique to each isoform. These results enhance our current knowledge of pectinase specificity and processivity and contribute to the development of more efficient pectinase usage within many industries.