Structural and funcional studies of the enzymes SsuD and SsuE from the alkanesulphonate ABC transporter and the periplasmic binding protein (PbP) from Xanthomonas axopodis pv.citri

Author(s)

Vanessa Rodrigues Pegos

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2011-01-25

Examining board members

Andrea Balan; Celso Eduardo Benedetti; Luis Eduardo Soares Netto

• Advisor: Andrea Balan

Abstract

Sulfur uptake in Escherichia coli is dependent of the ABC transporter SbpCysAWD (ATP Binding Cassete), which belongs to a regulon with 26 genes. In the sulfate absence, the bacteria induces the expression of two other ABC transporters, for alkanesulfonates (SsuABCDE) and aliphatic sulfonates (TauABCDE), which are responsible for the uptake and oxidation of these compounds to sulfite and aldehyde. Although its functionality has been not showed, Xanthomonas axonopodis pv. citri has all the genes involved in this regulon, including the alkanesulfonate transporter and enzymes. In order to continue the characterization of this transport, this work shows for the first time, the structural and functional characterization of the proteins SsuD and SsuE. Biochemical analyses associated to the bioinformatics and molecular modeling tools revealed that the SsuD and SsuE form a two-components system, where SsuD is the oxidoreductase responsible for the NAD(P)H oxidation followed by the FMN reduction. The protein was expressed and purified from E. coli cells with a molecular mass of the 39 kDa and it is organized in a octamer, such was demonstrated by the small angle scattering X-ray and ultracentrifugation assays. The tri-dimensional model of SsuD reveals a TIM barrel folding and conservation of residues that allow the oligomerization and NADP interaction. Enzymatic assays showed a high affinity binding of SsuD and NADP (0,21 µM) and that the protein was able to obtain the cinetic parameters, such as Km: 0.1877µM -1; Kcat: 7,192 s-1; Vmáx: 0.7911 µM/min; Kcat/Km: 3,8x 10-7 m 1S-1. Indeed, SsuD crystals were obtained in different conditions. The work still shows the charactrization of the Pbp protein, from X. axonopodis, believed to be the fosfonate/fosfate binding protein. Pbp was expressed with a molecular mass of 33 kDa, and spectroscopic analyses revealed conformational changes at the secondary structure content in presence of fosfonates, as well as an increased thermal stability in presence of spermidine, which was used for the crystallization trials. Crystals were obtained but still not tested. All results presented in this work can bring some light to the strategies that X. axonopodis uses for growth and infection and they will direct our studies for laboratorial and in vivo analyses (AU)

• FAPESP's process: 08/07676-1 - Structural and functional studies of the enzymes SsuD and SsuE of the alkane sulfonate transport system of Xanthomonas axonopodis pv. citri
• Grantee: Vanessa Rodrigues Pegos
• Support Opportunities: Scholarships in Brazil - Master