Structural and functional studies of the enzyme N5, N10 -methylenetetrahydrofolate dehydrogenase-cyclohydrolase of Xanthomonas albilineans applied to the discovery of inhibitor candidates for the treatment of leaf scald

Author(s)

Renata Vieira Bueno

• Document type: Doctoral Thesis
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT)
• Defense date: 2018-04-05

Examining board members

Rafael Victório Carvalho Guido; Glaucius Oliva; Luis Octavio Regasini; Flávio Henrique da Silva; Daniela Barretto Barbosa Trivella

• Advisor: Rafael Victório Carvalho Guido

Abstract

The leaf scald disease is a severe condition which affects sugarcane crops. Leaf scald is caused by the Gram-negative bacteria Xanthomonas albilineans and it has a dramatic impact on crop productivity, including the yield reduction and dropping the quality of the juice. The impact of this disease besides the absence of chemical or biological agents to treat it stimulates the research towards the discovery and development of bioactive molecules as lead candidates to new agrochemicals. In this context, the enzyme N5, N10-methylenetetrahydrofolate dehydrogenase-cyclohydrolase (XaFolD) stands out as a potential molecular target. Facing this scenario, the PhD project comprised the structural and functional characterization of X. albilineans FolD (XaFolD) and the screening of molecules to identify inhibitors. For this purpose, integrated studies in structural molecular biology and biological chemistry have been carried out. Suitable protein for crystallization and kinetic assays was produced by heterologous expression. The XaFolD dehydrogenase and cyclohydrolase activities were characterized, revealing KM values of 50 ± 10 µM for N5, N10- methylenetetrahydrofolate, 32 ± 3 µM for N5, N10-methenyltetrahydrofolate, and 688 ± 81 µM for NADP+. The structure of XaFolD in the apo form was elucidated at 2.1 Å resolution and it revealed a protein consisting of 11 alpha helices and 9 beta sheets distributed in the catalytic N-terminal domain and the C-terminal dinucleotide binding domain. Screening of 1,124 fragments by biolayer interferometry (BLI) revealed three ligands, DDD00808259, DDD01305586 and DDD00100784, with determined KD values of 260 µM, 3 mM and 210 M, respectively. Furthermore, a virtual screening was performed to identify XaFolD ligands. As a result, 31 chemically diverse compounds were selected to be evaluated by potency assays. Additionally, phenotypical assays against X. albilineans have been performed and inhibitors were identified. Among them, the compound THP2 presented the highest potency, with a determined value of EC50 equal to 23 ± 2 µM. The structural, kinetic, and biophysical data obtained in this PhD thesis provide the molecular basis for discovering and planning bioactive molecules as agrochemicals to control leaf scald disease. (AU)

• FAPESP's process: 13/04737-8 - Discovery and Design of Folate Synthesis Inhibitors as Agrochemicals Candidates for Sugar Cane Culture
• Grantee: Renata Vieira Bueno
• Support Opportunities: Scholarships in Brazil - Doctorate