title: Directed evolution induces tributyrin hydrolysis in a virulence factor of Xylella fastidiosa using a duplicated gene as a template.
abstract: Duplication of genes is one of the preferred ways for natural selection to add advantageous functionality to the genome without having to reinvent the wheel with respect to catalytic efficiency and protein stability. The duplicated secretory virulence factors of Xylella fastidiosa (LesA, LesB and LesC), implicated in Pierce's disease of grape and citrus variegated chlorosis of citrus species, epitomizes the positive selection pressures exerted on advantageous genes in such pathogens. A deeper insight into the evolution of these lipases/esterases is essential to develop resistance mechanisms in transgenic plants. Directed evolution, an attempt to accelerate the evolutionary steps in the laboratory, is inherently simple when targeted for loss of function. A bigger challenge is to specify mutations that endow a new function, such as a lost functionality in a duplicated gene. Previously, we have proposed a method for enumerating candidates for mutations intended to transfer the functionality of one protein into another related protein based on the spatial and electrostatic properties of the active site residues (DECAAF). In the current work, we present in vivo validation of DECAAF by inducing tributyrin hydrolysis in LesB based on the active site similarity to LesA. The structures of these proteins have been modeled using RaptorX based on the closely related LipA protein from Xanthomonas oryzae. These mutations replicate the spatial and electrostatic conformation of LesA in the modeled structure of the mutant LesB as well, providing in silico validation before proceeding to the laborious in vivo work. Such focused mutations allows one to dissect the relevance of the duplicated genes in finer detail as compared to gene knockouts, since they do not interfere with other moonlighting functions, protein expression levels or protein-protein interaction.
authors.affiliation: Plant Sciences Department, University of California, Davis, CA, 95616, USA.
authors.forename: Hossein
authors.initials: H
authors.lastname: Gouran
authors.affiliation: Plant Sciences Department, University of California, Davis, CA, 95616, USA.|Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai, 400 005, India.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Chakraborty
authors.affiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai, 400 005, India.
authors.forename: Basuthkar J
authors.initials: BJ
authors.lastname: Rao
authors.affiliation: Science Institute, Department of Biochemistry, University of Iceland, Dunhaga 3, IS-107 Reykjavik, Iceland.
authors.forename: Bjarni
authors.initials: B
authors.lastname: Asgeirsson
authors.affiliation: Plant Sciences Department, University of California, Davis, CA, 95616, USA.
authors.forename: Abhaya
authors.initials: A
authors.lastname: Dandekar