GENERAL RESEARCH INTERESTS
Bacterial physiology
Membrane protein biochemistry
Protein-protein interactions
Hydrogen metabolism
Enzyme engineering
Synthetic biology
CURRENT PROJECTS
In prokaryotes, generation of energy by respiratory electron transfer chains involves the plasma membrane. One striking feature of Escherichia coli and Salmonella physiology is a flexible respiratory metabolism which stems from an elaborate bank of membrane-associated respiratory enzymes. Many respiratory enzymes are located in the cell membranes and are often extremely complex consisting of multiple subunits and associated redox cofactors. A subset of exported proteins 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 by an export apparatus termed the twin-arginine translocation (Tat) system.
Understanding bacterial hydrogen metabolism
‘Biohydrogen’ is the production, or consumption, of molecular hydrogen by living organisms and is a process central to the physiology of many microbes, including green algae, strict anaerobes, soil aerobes, and human pathogens such as Helicobacter and Salmonella. Hydrogen has the highest energy per weight of any fuel and is an important feedstock for the chemical industry; most importantly, biohydrogen offers the prospect of fully renewable hydrogen, freed from any dependence on fossil fuel. Enteric bacteria (e.g. Escherichia coli) evolve hydrogen when cultured under fermentative conditions, i.e. with glucose and in the absence of all other respiratory electron acceptors. E. coli has become well established as a ‘chassis’ organism for biotechnologists interested in genetically modifying the cell’s metabolism, or even designing completely synthetic activities.
Hydrogenases are extremely active metalloenzymes responsible for the vast majority of microbial hydrogen evolution. The two main classes of hydrogenase, which share no sequence homology, are known as [FeFe] and [NiFe] according to the metals in the active site. In both cases, the active site Fe is further coordinated by CO and CN- ligands. E. coli contains only [NiFe]hydrogenases and of these, Hydrogenases-1 and -2 (Hyd-1 and Hyd-2) exist minimally as heterodimers of a ‘large’ subunit, which contains the [NiFe] active site, and a ‘small’ subunit, which contains three [Fe-S] clusters. Indeed, all of the best characterised [NiFe]hydrogenases follow this design. Hyd-1 and Hyd-2 are located at the periplasmic side of the membrane where they participate in respiratory hydrogen oxidation linked to quinone reduction (or hydrogen 'uptake'). Neither enzyme has a role to play during in vivo hydrogen evolution. Instead, hydrogen evolution is catalysed solely by hydrogenase-3 (Hyd-3), a component of the formate hydrogenlyase (FHL) complex. The E. coli genome also encodes a cryptic Hyd-3 homolog termed Hyd-4.
We have developed the tools to isolate the FHL complex and are busy trying to understand its structure and function.
Assembly of bacterial hydrogenases
under construction
Engineering hydrogenases and bacterial hydrogen metabolism
Hydrogen is a potential ‘clean fuel’ of the future and fermentative bacteria like Escherichia coli can generate large amounts of molecular hydrogen at ambient temperature thanks to their remarkable hydrogenase enzymes. We hope to engineer a ‘Superbug’ maximized for bio-hydrogen production.
The Tat Proofreading Process
The central dogma of Tat transport is that Tat substrates are fully folded before export, and as a result it is essential that biosynthesis and assembly of complex respiratory enzymes is completed in the cytoplasm before the final Tat transport event is even attempted. Recent work has unearthed a cellular mechanism that prevents premature targeting of Tat signal-bearing proteins until all biosynthetic processes are concluded. The ‘Tat proofreading’ process involves the pre-export interaction of Tat signal peptides with dedicated binding proteins, or ‘chaperones’. The paradigm Tat proofreading chaperones are TorD and NapD, small multi-functional proteins that binds tightly to specific Tat signal peptides thus preventing premature export. Ligand binding by purified TorD and NapD, as well as a wide range of other TorD-like proteins, is studied in my group by techniques including isothermal titration calorimetry (ITC), electron spin resonance (ESR) and protein crystallography.