258 - Studies of Disulfide Reductase Activity and Phylogenetic Distribution of Xylella Fastidiosa YbbN Protein

Abstract

Xylella fastidiosa is a gram-negative bacteria and a plant pathogen that causes Citrus Variegated Chlorosis (CVC) in Brazil; Pierce´s disease in grapevines and Olive Quick Decline Syndrome in Southern Italy. In the infection process, the generation of oxidizing species in the extracellular environment is a major defense mechanism used by the plant. To counteract this oxidative insult imposed by the host, pathogen contains a complex array of defense systems that include antioxidant enzymes, like members of thioredoxin (Trx) superfamily. YbbN is a 32 kDa protein that possesses a N-terminal portion (12 kDa) Trx domain and two TPR (tetratricopeptide repeat) domains (20 kDa) in the C-terminal portion of the enzyme with still unknown functions. The Trx-domain of YbbN from X. fastidiosa has the common CXXC motif, in this case composed of CAPC residues, while most Trx enzymes contain the CGPC motif. In contrast, YbbN from E. coli possesses a SXXC motif and is devoid of thiol-disulfide reductase activity. Here, we describe the disulfide reductase activity of YbbN from X. fastidiosa and analyzed the phylogenetic distribution of the protein compared to others YbbN proteins. The disulfide reductase activity was measured by the DTNB reduction assay and the phylogenetic distribution of YbbN enzymes were made using Jackhmmer to find homologues sequences, CD-HIT to cluster homologue sequences, PRANK to align sequences and MEGA7 to construct the tree with maximum likelihood estimation. Three major groups were identified: the canonical CXXC active site group that includes the X. fastidiosa YbbN; a group with SXXC active site that includes the E. coli YbbN protein; and the least representative group with QXXC active site. Biochemical and structural characterization of X. fastidiosa YbbN is underway and the specific activity of DTNB reduction by YbbN was determined as 13,22 μMol.s -1 .mg -1 . We also found that X. fastidiosa TrxR was capable of reducing YbbN at NADPH expense. Further enzymatic analyses are in progress that include the investigation of the possible role of YbbN as substrate for Cys-based peroxidases such as Ohr , PrxQ and AhpC. Acknowledgements Fapesp, CEPID Redoxoma, CNPq, CAPES.