Assembly and targeting of complex bacterial enzymes

Research

 

Work in this research group concentrates on the molecular biology of bacterial respiration. Escherichia coli and Salmonella enterica are related bacteria that  display versatile metabolic capabilities. These bacteria can grow easily in the absence of oxygen either by substituting a number of different organic or inorganic compounds as electron acceptors, or by switching to a fermentative metabolism. This respiratory flexibility is thanks to a bank of different respiratory enzymes available to these bacteria.

Respiratory enzymes are complex enzymes comprising many protein subunits containing any number of prosthetic groups. Moreover, these enzymes are often buried in the cell membranes or located completely outside the cytoplasm. As a result, biosynthesis of complex respiratory enzymes requires the coordination of cofactor insertion, protein folding, and protein targeting processes. A subset of respiratory enzymes are synthesised as precursors with N-terminal signal peptides bearing a conserved SRRxFLK ‘twin-arginine’ amino acid motif. Proteins with twin-arginine signal peptides are transported fully folded by an export apparatus termed the twin-arginine translocation (Tat) system. Research in the group currently focuses on novel molecular, biochemical, and applied aspects of Tat-targeted bacterial respiratory enzymes.

In 2004, research in the group identified a chaperone-mediated ‘Tat proofreading’ process involved in the coordination of protein assembly and export. The paradigm Tat proofreading chaperone is TorD, a small multi-functional protein, which binds tightly to the Tat signal peptide of trimethylamine N-oxide reductase thus preventing premature export of the enzyme until all assembly proceses are complete. TorD-like chaperones are almost ubiquitous in bacteria and archaea and the group has been using genetic and biophysical approaches to understanding the chaperone-signal peptide interaction. TorD family proteins are not the only Tat proofreading chaperones in nature. In 2007, the group identified NapD as chaperone that bound tightly to the Tat signal peptide of nitrate reductase. The 3D solution structure of NapD was solved through collaboration with researchers at Radboud University Nijmegen, which revealed that NapD was completely unrelated to the TorD protein.

Understanding the biosynthetic processes required to build complex respiratory enzymes could have future applications. For example, under fermentative conditions a set of complex enzymes is produced that enables the bacterium to evolve hydrogen gas. Biological approaches to hydrogen production are growing in importance as fossil fuel resources verge on the limits of economical extraction, and the environmental impact of carbon emissions gains long-overdue recognition. In collaboration with researchers at the University of Oxford, the group has been studying the structure and function of bacterial hydrogenase enzymes

Teaching

Publications

 PUBLICATIONS (since 2008)

HYDROGENASES

Parkin,A., and Sargent,F. (2012) The hows and whys of aerobic hydrogen metabolism. Current Opinion in Chemical Biology 16: 26-34

Parkin,A., Bowman,L., Roessler,M.M., Davies,R.A., Palmer,T., Armstrong,F.A., and Sargent,F. (2012) How Salmonella oxidises H₂ under aerobic conditions. FEBS Letters 586, 536-544.

Pinske,C., McDowall,J.S., Sargent,F., and Sawers,R.G. (2012) Analysis of hydrogenase-1 levels reveals an intimate link between carbon and hydrogen metabolism in Escherichia coli K-12. Microbiology 158: 856-868.

Pinske,C., Krueger,S., Soboh,B., Ihling,C., Kuhns,M., Braussemann,M., Jaroschinsky,M., Sauer,C., Sargent,F., Sinz, A. and Sawers, R.G. (2011) Efficient electron transfer from hydrogen to benzyl viologen by the [NiFe]hydrogenases of Escherichia coli is dependent on the co-expression of the iron-sulfur cluster-containing small subunit. Archives of Microbiology 193: 893-903.

Lukey,M.J., Roessler,M.M., Parkin,A., Evans,R.M., Davies,R.A., Lenz, O., Friedrich,B., Sargent,F. and Armstrong,F.A. (2011) Oxygen-tolerant [NiFe] hydrogenases: the individual and collective importance of supernumerary cysteines at the proximal Fe-S cluster. Journal of the American Chemical Society 133: 16881-16892.

 Lukey,M.J., Parkin,A., Roessler,M.M., Murphy,B.J., Harmer,J., Palmer,T., Sargent,F., and Armstrong,F.A. (2010) How Escherichia coli is equipped to oxidize hydrogen under different redox conditions. Journal of Biological Chemistry 285, 3928-39238.

 Lazarus,O., Woolerton,T.W., Parkin,A., Lukey,M.J., Reisner,E., Seravalli,J., Pierce,E., Ragsdale,S.W., Sargent,F., and Armstrong,F.A. (2009) Water-gas shift reaction catalyzed by redox enzymes on conducting graphite platelets. Journal of the American Chemical Society 131, 14154-14155.

  BIOHYDROGEN & METAL BIOREMEDIATION

 Orozco,R.L., Redwood,M.D., Yong,P., Caldelari,I., Sargent,F., and Macaskie,L.E. (2010) Towards an integrated system for bio-energy: hydrogen production by Escherichia coli and use of palladium-coated waste cells for electricity generation in a fuel cell. Biotechnology Letters 32, 1837-1845.

 Deplanche,K., Caldelari,I., Mikheenko,I.P., Sargent,F., and Macaskie,L.E. (2010) Involvement of hydrogenases in the formation of highly catalytic Pd(0) nanoparticles by bioreduction of Pd(II) using Escherichia coli mutant strains. Microbiology 156, 2630-2640.

 Yong,P., Mikheenko,I.P., Deplanche,K., Sargent,F., and Macaskie,L.E. (2009) Biorecovery of precious metals from wastes and conversion into fuel cell catalyst for electricity production. Advanced Materials Research 71-73, 729-732.

 Redwood,M.D., Mikheenko,I.P., Sargent,F., and Macaskie,L.E. (2008) Dissecting the roles of E. coli hydrogenases in biohydrogen production. FEMS Microbiology Letters 278, 48-55.

 TAT PROOFREADING CHAPERONES

 Grahl,S., Maillard,J., Spronk,C.A., Vuister,G.W., and Sargent,F. (2012) Overlapping transport and chaperone-binding functions within a bacterial twin-arginine signal peptide. Molecular Microbiology 83,1254-1267.

Coulthurst,S.J., Dawson, A., Hunter,W.N., and Sargent,F. (2012) Conserved signal peptide recognition systems across the prokaryotic domains. Biochemistry 51, 1678-1686.

Guymer,D., Maillard,J., Agacan, M.F., Brearley,C.A., and Sargent,F. (2010) Intrinsic GTPase activity of a bacterial twin-arginine translocation proofreading chaperone induced by domain swapping. The FEBS Journal 277, 511-525.

 Ize,B., Coulthurst,S.J., Hatzixanthis,K., Caldelari,I., Buchanan,G., Barclay,E.C., Richardson,D.J., Palmer,T., and Sargent,F. (2009) Remnant signal peptides on non-exported enzymes: implications for the evolution of prokaryotic respiratory chains. Microbiology 155, 3992-4004.

 Guymer,D., Maillard,J., and Sargent,F. (2009) A genetic analysis of in vivo selenate reduction by Salmonella enterica serovar Typhimurium LT2 and Escherichia coli K12. Archives of Microbiology 191, 519-28.

 Buchanan,G., Maillard,J., Nabuurs,S.B., Richardson,D.J., Palmer,T., and Sargent,F. (2008) Features of a twin-arginine signal peptide required for recognition by a Tat proofreading chaperone. FEBS Letters 582, 3979-3984.

 Lüke,I., Butland,G., Moore,K., Buchanan,G., Lyall,V., Fairhurst,S.A., Greenblatt,J.F., Emili,A., Palmer,T., and Sargent,F. (2008) Biosynthesis of the respiratory formate dehydrogenases from Escherichia coli: characterization of the FdhE protein. Archives of Microbiology 190, 685-696.

 THE BACTERIAL TAT PATHWAY

 Maldonado,B., Kneuper,H., Buchanan,G., Hatzixanthis,K., Sargent,F., Berks,B.C., and Palmer,T. (2011) Characterisation of the membrane-extrinsic domain of the TatB component of the twin-arginine protein translocase. FEBS Letters 585, 478-84.

 Palmer,T., Berks,B.C., and Sargent F. (2010) Analysis of Tat targeting function and twin-arginine signal peptide activity in Escherichia coli. Methods in Molecular Biology 619, 191-216.

 Lüke,I., Handford,J.I., Palmer,T., and Sargent,F. (2009) Proteolytic processing of Escherichia coli twin-arginine signal peptides by LepB. Archives of Microbiology 191, 919-925.

 Tarry,M., Arends,S.J., Roversi,P., Piette,E., Sargent,F., Berks,B.C., Weiss,D.S., and Lea,S.M. (2009) The Escherichia coli cell division protein and model Tat substrate SufI (FtsP) localizes to the septal ring and has a multicopper oxidase-like structure. Journal of Molecular Biology 386, 504-519.

 Caldelari,I., Palmer,T., and Sargent,F.(2008) Escherichia coli tat mutant strains are able to transport maltose in the absence of an active malE gene. Archives of Microbiology 189, 597-604.