title: Laboratory evolution of Escherichia coli thioredoxin for enhanced catalysis of protein oxidation in the periplasm reveals a phylogenetically conserved substrate specificity determinant.
abstract: Thioredoxin exported into the Escherichia coli periplasm catalyzes the oxidation of protein thiols in a DsbB-dependent function. However, the oxidative activity of periplasmic thioredoxin is insufficient to render dsbA(-) cells susceptible to infection by M13, a phenotype that is critically dependent on disulfide bond formation in the cell envelope. We sought to examine the molecular determinants that are required in order to convert thioredoxin from a reductant into an efficient periplasmic oxidant. A genetic screen for mutations in thioredoxin that render dsbA(-) cells sensitive to infection by M13 led to the isolation of a single amino acid substitution, G74S. In vivo the TrxA(G74S) mutant exhibited enhanced catalytic activity in the oxidation of alkaline phosphatase but was unable to oxidize FlgI and restore cell motility. In vitro studies revealed that the G74S substitution does not affect the redox potential of the thioredoxin-active site or its kinetics of oxidation by DsbB. Thus, the gain of function afforded by G74S stems in part from its altered substrate specificity, which also rendered the protein more resistant to reduction by DsbD/DsbC in the periplasm.
authors.affiliation: Department of Chemical Engineering, University of Texas, Austin, USA.
authors.forename: Lluis
authors.initials: L
authors.lastname: Masip
authors.forename: Daniel
authors.initials: D
authors.lastname: Klein-Marcuschamer
authors.forename: Shu
authors.initials: S
authors.lastname: Quan
authors.forename: James C A
authors.initials: JC
authors.lastname: Bardwell
authors.forename: George
authors.initials: G
authors.lastname: Georgiou
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D017124:Conserved Sequence; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005838:Genotype; D005998:Glycine / Q000032:analysis; D006629:Hirudins / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010802:Phylogeny; D010957:Plasmids; D011487:Protein Conformation; D013379:Substrate Specificity; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D006629:Hirudins; D013879:Thioredoxins; D000469:Alkaline Phosphatase; D005998:Glycine