Functional characterization of unique enzymes in Xanthomonas euvesicatoria related to degradation of arabinofurano-oligosaccharides on hydroxyproline-rich glycoproteins

Abstract

In this study, we clarified the functions of three uncharacterized enzymes, XCV2724, XCV2728, and XCV2729, in Xanthomonas euvesicatoria, the causal agent of bacterial spot of tomato and pepper. The genes corresponding to the three enzymes are homologs of hypBA1, hypBA2, and hypAA from Bifidobacterium longum and are unique to Xanthomonas spp. among plant pathogenic bacteria. Functional characterization of the recombinant enzymes expressed using microbial systems revealed that they degrade the arabinofurano-oligosaccharides present on hydroxyproline (Hyp)-rich glycoproteins (HRGPs) such as extensin and solanaceous lectins in plant cell walls. These enzymes work coordinately to degrade the oligosaccharides. First, XeHypAA (XCV2728), belonging to the glycoside hydrolase (GH) 43 family, releases L-arabinose from L-arabinofuranose (Araf)-α1,3-Araf-ß1,2-Araf-ß1,2-Araf-ß-Hyp (Ara4-Hyp), cleaving its α1,3 bond; second, XeHypBA2 (XCV2729), belonging to the GH121 family, releases the disaccharide Araf-ß1,2-Araf from Araf-ß1,2-Araf-ß1,2-Araf-ß-Hyp (Ara3-Hyp); finally, XeHypBA1 (XCV2724), belonging to GH family 127, releases L-arabinose from Araf-ß-Hyp (Ara-Hyp). In summary, the main oligosaccharide structure of Ara4-Hyp on the HRGPs is degraded to Ara3-Hyp, then to Ara-Hyp, and finally to Ara monosaccharides by the action of these three enzymes. HRGPs containing oligosaccharide substrates have been reported to contribute to plant defense, and interestingly, the promoter region of the operon (xehypBA2 and xehypAA) contains the plant-inducible promoter box for binding the regulator protein HrpX involved in pathogenicity. We then analyzed the expression level of the operon gene in hrp-inducing medium and in plants and constructed gene-deletion mutants. However, although the operon was evidently upregulated by HrpX, three single-gene deletion mutants (ΔxehypBA1, ΔxehypBA2, ΔxehypAA) and even a triple-gene deletion mutant (ΔxehypBA1-BA2-AA) remained pathogenic, and had no effect on nonhost resistance, either, indicating that these three enzymes are not involved in either pathogenicity or nonhost resistance reactions. This is the first report of enzymes in plant pathogenic bacteria that catalyze the degradation of Hyp-linked-L-arabinofuranosides in plant cell walls.

Crystal structure of β-L-arabinobiosidase belonging to glycoside hydrolase family 121

“…Bifidobacteria are representative species of gut microbes that are beneficial to human health while Xanthomonas species contain pathogens that cause bacterial spots on a wide variety of plant species. The genes for β-L-arabinooligosaccharide-degradation enzymes in X. euvesicatoria (xehypBA1-BA2-AA) are not involved in either pathogenicity or non-host resistance reactions [42]. However, the distribution of GH121 genes suggests that bifidobacteria and Xanthomonas species possibly benefit from the disaccharide-releasing enzyme and the disaccharide-specific importer system on their biological niches in order to live on special types of plant glycans, such as Ara 3 -Hyp β-L-arabinooligosaccharides on HRGPs.…”

“…186 sequences from Xanthomonas species formed a very large group with little or no sequence diversity, 175 of them being in a zero branch between them. A HypBA2 homolog from Xanthomonas euvesicatoria (XeHypBA2) [42] was also included in this cluster. The full amino acid sequence alignment revealed that the amino acid residues at the Ca2 site are essentially conserved in GH121, whereas those at Ca1 and Ca3 are not conserved (S3 Fig) . Sequence logos around the three acidic residues (E383, D515, and E713, Fig 4B) illustrated that all of them are completely conserved in GH121, and nearby sequences are also highly conserved.…”

Cracking the “Sugar Code”: A Snapshot of N- and O-Glycosylation Pathways and Functions in Plants Cells

“…Several GHs from different bacterial sources have been described that hydrolyze specific linkages within O-glycans of EXTs (Table 2). The GH127 enzyme from Xanthomonas euvesicatoria XeHypBA1 was described as a β-L-(1 → 2)arabinofuranosidase (Nakamura et al, 2018) while two GH121 members, one from Bifidobacterium bifidum HypBA2 and XeHypBA2, were shown to hydrolyze the β-L-Araf -(2 → 1) linkages (Fujita et al, 2011(Fujita et al, , 2014Nakamura et al, 2018). (Nakamura et al, 2018) (Table 2 and Figure 2).…”

Structural analysis of β‐L‐arabinobiose‐binding protein in the metabolic pathway of hydroxyproline‐rich glycoproteins in Bifidobacterium longum

“…Our preliminary study indicated that transcription levels of the b-arabinooligosaccharide utilization genes (BLLJ_0208, BLLJ_0211, BLLJ_0212, and BLLJ_0213) were upregulated when B. longum JCM 1217 was grown with b-Ara 2 as the sole carbon source, but not with L-arabinose (data not shown). The xehypBA2-AA operon in X. euvesicatoria (plant pathogen) notably contains homologous genes of HypAA, HypBA2, and HypBA1 but does not have transporter genes [24]. The uptake system specific to b-Ara 2 may be specific to and required by these bifidobacterial species to effectively utilize carbon sources from plants in competition with other bacteria.…”

“…We previously reported structural and functional characterizations of HypBA1 [22,23] and a similar enzymatic degradation system from the plant pathogenic bacterium Xanthomonas euvesicatoria [24]. Here, we aimed to determine the three-dimensional structure and molecular and mechanistic functions of the SBP (BLLJ_0208) using X-ray crystallography, thermal shift assays, isothermal titration calorimetry (ITC), and molecular dynamics (MD) simulation.…”