Jonathan Beckwith

Department of Microbiology and Molecular Genetics
Harvard Medical School
200 Longwood Avenue
Boston, MA 02115
Tel: (617) 432-1920
Fax: (617) 738-7664
Email: jbeckwith@hms.harvard.edu
Web Page: The Beckwith Lab Page
4 postdoctoral fellows, 4 graduate students

We use genetics, biochemistry and bioinformatics to study the properties and evolution of enzyme systems in bacteria that are important for protein folding, protein translocation and responses to oxidative stress. For these studies, we are defining the pathways of electron transfer that confer a reducing environment on the cytoplasm and an oxidizing environment on extra-cytoplasmic compartments.

The glutathione/glutaredoxin and thioredoxin pathways of E. coli are important for the maintenance of the cysteines of cytoplasmic proteins in the reduced state and for the cell’s response to oxidative stress. We have isolated suppressor mutations that restore growth to cells mutated in these pathways. This allows us to evolve E. coli so that it uses alternative pathways for reducing cytoplasmic proteins. These suppressors include a remarkable conversion of a peroxidase into a disulfide reductase revealing the unusual plasticity of this protein, as well as a mutation of an enzyme involved in metabolism of the small sulfhydryl-containing molecule, lipoic acid. Further studies will focus on the effects of the mutations on protein structure, the impact on the oxidative state of the cytoplasm, and details of the new electron transfer pathways. We are also surveying a wide array of bacteria species for the presence of these alternative pathways by looking for altered versions of the genes for these enzymes that resemble our suppressor mutations.

In the periplasm of many bacteria, the DsbA and DsbB enzymes assist the folding of proteins by introducing disulfide bonds into them. By following the kinetics of disulfide bond formation in substrate proteins as they are translocated across the cytoplasmic membrane, we can take “snapshots” of the protein as it folds in vivo and determine what other cellular factors and features of the protein and its translocation influence the folding pathway. We study the mechanisms by which the enzymes DsbC and DsbD correct proteins that are misfolded as a result of formation of incorrect disulfide bonds. We also have developed a bioinformatic approach that allows us to analyze the genomes of hundreds of bacterial species to determine 1)whether they make disulfide bonds in their proteins and 2)whether different systems for carrying out these processes exist. This analysis led to the discovery that many bacteria have a previously unrecognized enzyme required for disulfide bond formation that is a homologue of vitamin K epoxide reductase, a mammalian protein involved in blood clotting.

References:

• Kadokura, H., Tian, H., Zander, T., Bardwell, J.C.A. and Beckwith, J. Snapshots of DsbA in action: detection of proteins in the process of oxidative folding. Science 303:534-537 (2004).
• Cho, S.-H., Porat, A., Ye, J., and Beckwith, J. Redox-active cysteines of a membrane electron transporter DsbD show dual compartment accessibility. EMBO J. 8:3509-3520 (2007).
• Faulkner, M.J., Veeravalli, K., Gon, S., Georgiou, G., and Beckwith, J. Functional plasticity of a peroxidase allows evolution of diverse disulfide-reducing pathways.
  Proc. Natl. Acad. Sci. U.S.A. 105:6735-40 (2008).
• Dutton, R.J., Boyd, D., Berkmen. M., and Beckwith, J. Bacterial species exhibit diversity in their mechanisms and capacity for protein disulfide bond formation. Proc. Natl. Acad. Sci., U.S.A. (in press).