Insights into the Activity and Substrate Binding of Xylella fastidiosa Polygalacturonase by Modification of a Unique QMK Amino Acid Motif Using Protein Chimeras

Abstract

Polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa possesses a single polygalacturonase gene, pglA (PD1485), and X. fastidiosa mutants deficient in the production of polygalacturonase are non-pathogenic and show a compromised ability to systemically infect grapevines. These results suggested that grapevines expressing sufficient amounts of an inhibitor of X. fastidiosa polygalacturonase might be protected from disease. Previous work in our laboratory and others have tried without success to produce soluble active X. fastidiosa polygalacturonase for use in inhibition assays. In this study, we created two enzymatically active X. fastidiosa / A. vitis polygalacturonase chimeras, AX1A and AX2A to explore the functionality of X. fastidiosa polygalacturonase in vitro. The AX1A chimera was constructed to specifically test if recombinant chimeric protein, produced in Escherichia coli, is soluble and if the X. fastidiosa polygalacturonase catalytic amino acids are able to hydrolyze polygalacturonic acid. The AX2A chimera was constructed to evaluate the ability of a unique QMK motif of X. fastidiosa polygalacturonase, most polygalacturonases have a R(I/L)K motif, to bind to and allow the hydrolysis of polygalacturonic acid. Furthermore, the AX2A chimera was also used to explore what effect modification of the QMK motif of X. fastidiosa polygalacturonase to a conserved RIK motif has on enzymatic activity. These experiments showed that both the AX1A and AX2A polygalacturonase chimeras were soluble and able to hydrolyze the polygalacturonic acid substrate. Additionally, the modification of the QMK motif to the conserved RIK motif eliminated hydrolytic activity, suggesting that the QMK motif is important for the activity of X. fastidiosa polygalacturonase. This result suggests X. fastidiosa polygalacturonase may preferentially hydrolyze a different pectic substrate or, alternatively, it has a different mechanism of substrate binding than other polygalacturonases characterized to date.

Root infection and aerial colonization of eucalypt host plants by Erwinia psidii

“…For instance, E. amylovora has an arsenal of enzymes that degrade cell walls and are important for virulence (Zhao et al 2005). Also, X. fastidiosa is able to degrade the xylem vessels due to the presence of a gene encoding an endopolygalacturonase (Warren et al 2015). Research aimed at studying the role of cell-wall degrading enzymes for the interaction of E. psidii with eucalypt host plants could help better understand xylem colonization.…”

Functional and Evolutionary Characterization of a UDP-Xylose Synthase Gene from the Plant Pathogen Xylella fastidiosa, Involved in the Synthesis of Bacterial Lipopolysaccharide

“…Functional assays performed with recombinant X. fastidiosa proteins have also been employed to gather information regarding the structure and enzymatic properties of a few elements, potentially involved in bacterial resistance, adaptation, and virulence, such as heat/cold shock proteins, oxidative stress resistance factors, and the polygalacturonase gene (believed to contribute to bacterial dissemination within xylem vessels), among others. − Moreover, because the occlusion of xylem by bacterial aggregates is considered one of the main factors associated with X. fastidiosa -related diseases, functional analyses have also attempted to characterize outer membrane adhesins and enzymes involved in the production of glycans, such as EPS and LPS, which have been shown to participate in biofilm formation and bacterial adherence. − …”

Screening of Sugarcane Proteins Associated with Defense against Leifsonia xyli subsp. xyli, Agent of Ratoon Stunting Disease

“…Endo-PGs, encoded by the pglA gene, are essential factors for plant pathogens to colonize the host and degrade pectin in the plant cell walls, resulting in the maceration of host tissues [13,14]. The pglA gene has been proved to be a pathogenicity gene in Pseudomonas solanacearum [15], Xylella fastidiosa [16], and Xanthomonas oryzae pv. oryzae [17].…”