Characterization of FK506-binding protein of XopAZ from Xanthomonas cynarae
Abstract
Xanthomonas cynarae is a species of proteobacteria that causes a bacterial disease in artichokes. By injecting avirulence proteins (Avr protein) into the host cell through the Type III Secretion System (T3SS), X. cynarae causes brown spots on artichoke leaves which could lead to financial loss for farmers due to its visual appearance. XopAZ is a protein that is produced in X. cynarae that we hypothesize is responsible of refolding of the Avr protein injected by the bacterium inside the plant host cell. We have determined that XopAZ is homologous to the Escherichia coli sensitive to lysis protein D (SlyD) (70% amino acid identity) and with the E. coli surface layer protein A (SlpA) (50% amino acid identity). Both SlpA and SlyD are known to contain two domains: a chaperone/insert-in-flap domains (IF domain) and a prolyl isomerase domain (PPIase/ FKBP domain). The presence of the IF domain helps to fold the unfolded or misfolded protein correctly; the FKBP domain catalyzes the isomerization of peptide bonds between an amino acid and proline. In a previous study, hexahistidine-tagged XopAZ was expressed in E. coli and purified by Nickel (Ni2+) Chelate Affinity Chromatography. The IF domain activity of purified XopAZ were measured via lysozyme aggregation assays and citrate synthase aggregation assays. However, the results of both studies showed no IF activity for XopAZ. In this study, we describe the preparation of and results from a FK506 binding assay to determine if XopAZ has an FKBP activity.
Characterization of FK506-binding protein of XopAZ from Xanthomonas cynarae
Xanthomonas cynarae is a species of proteobacteria that causes a bacterial disease in artichokes. By injecting avirulence proteins (Avr protein) into the host cell through the Type III Secretion System (T3SS), X. cynarae causes brown spots on artichoke leaves which could lead to financial loss for farmers due to its visual appearance. XopAZ is a protein that is produced in X. cynarae that we hypothesize is responsible of refolding of the Avr protein injected by the bacterium inside the plant host cell. We have determined that XopAZ is homologous to the Escherichia coli sensitive to lysis protein D (SlyD) (70% amino acid identity) and with the E. coli surface layer protein A (SlpA) (50% amino acid identity). Both SlpA and SlyD are known to contain two domains: a chaperone/insert-in-flap domains (IF domain) and a prolyl isomerase domain (PPIase/ FKBP domain). The presence of the IF domain helps to fold the unfolded or misfolded protein correctly; the FKBP domain catalyzes the isomerization of peptide bonds between an amino acid and proline. In a previous study, hexahistidine-tagged XopAZ was expressed in E. coli and purified by Nickel (Ni2+) Chelate Affinity Chromatography. The IF domain activity of purified XopAZ were measured via lysozyme aggregation assays and citrate synthase aggregation assays. However, the results of both studies showed no IF activity for XopAZ. In this study, we describe the preparation of and results from a FK506 binding assay to determine if XopAZ has an FKBP activity.
