Hydrogen Metabolism in Escherichia coli and Salmonella
Oxygen-tolerant Hydrogenases and their Biosynthesis
[NiFe]-hydrogenases are widespread in the microbial world and generally comprise a minimal functional unit of a large subunit, molecular weight ~ 60 kDa, which contains the [NiFe] active-site, and a small subunit, of ~ 35 kDa, which contains the Fe-S electron-transfer relay. Typically, [NiFe]-hydrogenase small subunits contain three Fe-S clusters: a distal [4Fe-4S] cluster at the surface of the protein and furthest from the [NiFe] active site; a medial [3Fe-4S] cluster; and a proximal Fe-S cluster, with variable properties, closest to the active site.
Several unusually oxygen-tolerant respiratory [NiFe] hydrogenases have been purified and spectroscopically and electrochemically characterized. These include the membrane bound hydrogenase (MBH) from R. eutropha and, from our own work, Hyd-1 and Hyd-5 from the enteric bacteria Escherichia coli and Salmonella, respectively. Recent work by several groups in this field have identified the unique proximal [4Fe-3S] cluster as having a key role in rescuing these enzymes from attack by oxygen. Structural, biochemical and molecular genetic approaches are being taken to understand the molecular basis of oxygen tolarance.
Tat proofreading - NapD Family Proteins
The napD gene has been known for many years since it is always genetically-linked to the napA gene, which encodes the periplasmic nitrate reductase. NapA-like nitrate reductases have been studied in great depth all over the world because of the central place of this enzyme in the global nitrogen cycle. It was also known for some time that the napD gene was “something to do with NapA biosynthesis”, though the details of exactly what were missing. It came as a surprise, therefore, when NapD turned out to be a signal peptide binding protein (Maillard et al. 2007 PNAS 104: 15641), since we had been fixated on the TorD family being the paradigm examples of such proteins. The high resolution NMR solution structure revealed a ferredoxin-like fold (PDB number 2jsx), which is very common and employed in ligand binding sites for a broad spectrum of biological processes.
NapD has a ferredoxin-like fold and a flexible C-terminus
In our most recent work we have been focused on understanding the NapD-NapA interaction at the molecular level. Further NMR approaches are beginning to reveal the structure of a chaperone-signal peptide complex for the first time.
The NapD chaperone in complex with the NapA signal peptide.
Tat proofreading - TorD Family Proteins
The largest, most widespread family of Tat proofreading chaperones belong to the TorD family, which contains hundreds of members across the bacterial and archaeal domains. TorD family proteins are also signal-peptide-binding-chaperones and available crystal structures show a helical fold arranged into two domains (N- and C-terminal) connected by a ‘hinge’. The proteins exist as monomers or domain-swapped homodimers where the N-domain of one protomer packs onto the C-domain of another. The hinge is often partially or completely unstructured and flexible, so many structures lack electron density for this region. This is a problem since the most highly conserved motif in TorD family proteins (EPxDH) is located within the hinge. TorD family proteins usually bind exclusively to one Tat signal peptide only, but the molecular basis of peptide selectivity, or the peptide binding-and-release mechanism, is not fully understood. The 3D structure of a signal peptide-chaperone complex is not available for any TorD family protein.
In our most recent work we have been focused on undertanding the function of a TorD family member from Archaeoblobus fulgidus. The Af0160 protein is apparently required for assembly of tetrathionate reductase in that organism - an enzyme that is important to the infection process of many pathogens, including Salmonella.
The A. fuldigus TtrD protein (formerly Af0160).