Laboratory Evolution of Escherichia coli Thioredoxin for Enhanced Catalysis of Protein Oxidation in the Periplasm Reveals a Phylogenetically Conserved Substrate Specificity Determinant*
- Lluis Masip‡,1,
- Daniel Klein-Marcuschamer‡,2,
- Shu Quan§,
- James C. A. Bardwell§ and
- George Georgiou‡¶∥,**,3
- Departments of ‡Chemical Engineering ¶Biomedical Engineering, and ∥Molecular Genetics and Microbiology and the **Institute for Cell and Molecular Biology, University of Texas, Austin, Texas 78712 and the §Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109
- ↵3 To whom correspondence should be addressed. E-mail: gg{at}che.utexas.edu.
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.
- Received June 22, 2007.
- Revision received October 24, 2007.
- The American Society for Biochemistry and Molecular Biology, Inc.
