Biochemical, structural and functional studies of glycosidic hydrolases from phytopatogen Xanthomonas citri subsp. citri str. 306

Author(s)

José Alberto Diogo

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2023-11-24

Examining board members

Mário Tyago Murakami; Claudio Chrysostomo Werneck; Cristina Elisa Alvarez Martinez; Wesley Cardoso Generoso

• Advisor: Mário Tyago Murakami; Priscila Oliveira de Giuseppe

Abstract

Brazil, as the second largest producer of citrus fruits in the world, has to deal with several diseases that attack this crop, including citrus canker, whose causal agent is the bacterium Xanthomonas citri subsp. citri. This phytopathogen has a high number of glycosyl hydrolases (GHs) encoded in its genome compared to other species of the genus, suggesting the use of different enzymes and plant cell wall deconstruction strategies during host colonization. Thus, the present work aimed to perform the biochemical, structural and functional characterization of five putative endoglucanases from "Xanthomonas citri" subsp citri str. 306 belonging to different GHs families. The enzymes XAC0028, XAC0029 and XAC0030 belong to the GH5_5 subfamily, the XAC0612 enzyme to the GH5_1 subfamily, and the XAC2522 to the GH9 family. Our results showed that these enzymes exhibit optimal activity in the pH range between 4.5 and 6.5, and optimal temperature between 30 and 40 °C, with the exception of XAC0612, which showed maximum activity at 65 °C. Activity assay on different polysaccharides revealed that the enzyme XAC0612 has high activity on glucomannan, higher than CMC, and can also hydrolyze (hetero)mannans. The structural characterization revealed that the active site of these endoglucanases are divergent such as the lack of aromatic side chains in the negative subsite region of XAC0029. Regarding kinetic behavior, the best values of the kcat/Km ratio on CMC were found for the enzymes XAC0612 (GH5_1) and XAC2522 (GH9) when compared to the GH5_5 enzymes. Using subcellular location predictors, associated with proteome data available in the literature, and also information about the activity of these enzymes, it was possible to propose their subcellular location. The enzymes XAC0028, XAC0029, XAC0612 are extracellular, the XAC0030 is periplasmatic and XAC2522 is associated with the outer membrane acting in the extracellular environment, which suggests the compartmentalization of the process of deconstruction of host cell wall components. The phenotypic evaluation of single knockouts of each of the enzymes shows that the action of the enzyme XAC0612 plays an important role in the deconstruction of glucomannan, ß-glucan and CMC by the pathogen, but its absence does not affect its ability to colonize and induce symptoms when inoculated into the host. Thus, in addition to the biochemical and structural characteristics of each of the enzymes studied, our results suggest that the sharing of substrates by them favors the survival of the microorganism and colonization of the host with consequent induction of citrus canker symptoms (AU)

• FAPESP's process: 18/03724-3 - Understanding the molecular system of degradation and utilization of cellulose by the phytopathogen Xanthomonas citri subsp. citri
• Grantee: José Alberto Diogo
• Support Opportunities: Scholarships in Brazil - Doctorate