Research
Current research
Research interests
The Biochemistry of Methylotrophs
Microbes that are able to grow on compounds with only one carbon atom [C1-compounds] such as methane, methanol, methylamine etc are called Methylotrophs. All the earlier work on these microbes is described in my book The Biochemistry of Methylotrophs.
My main interests are their carbon assimilation pathways and their energy transduction mechanisms. The first step in the oxidation of methanol is catalysed by the enzyme methanol dehydrogenase [MDH]. We first described this in 1964 and later showed it to be a new type of enzyme having a completely novel prosthetic group called PyrroloQuinoline Quinone [PQQ]; it was the first of a whole new family of enzymes called quinoproteins.
The PQQ-containing quinoproteins
These are the equivalent of flavoproteins that have the riboflavin derivatives FMN or FAD as their prosthetic groups. A major part of my work has been working out how energy [ATP] is obtained by linking the methanol dehydrogenase [MDH] in an electron transport chain to oxygen. After our discovery of PQQ [whose structure was later determined by others] it was found to be the prosthetic group of other bacterial enzymes such as glucose dehydrogenase [GDH] and we have also been involved in studies of this quinoprotein.
The structure and mechanism of the quinoprotein methanol dehydrogenase.
Our X-ray structure of methanol dehydrogenase was the first high resolution structure of a quinoprotein. It has an tetrameric structure. The α 2β 2-subunit (600 amino acids) is a superbarrel made up of eight radially-arranged β-sheets (the 'propeller fold'). PQQ, intimately bonded to a Ca2+ ion, is buried in the interior of the superbarrel. The floor of the active site chamber is formed by a tryptophan residue and the ceiling formed by a ring structure arising from a disulphide bridge between adjacent cysteine residues joined by a novel non-planar peptide bond. This is the only example of such a structure in an active enzyme; reduction inactivates (reversibly) the enzyme but its function is unknown. It has been proposed that a catalytic base (possibly Asp303) initiates the reaction by abstraction of a proton from the alcohol substrate. The Ca2+ ion is coordinated to The C-5 carbonyl oxygen of PQQ thus facilitating polarisation of the electrophilic C-5 for subsequent attack by an oxyanion or hydride.
The proposed role of PQQ as a vitamin
No mammalian PQQ-containing enzyme has been described. If such an enzyme does exist then it is very likely that PQQ will be a vitamin [analogous to riboflavin, needed in the diet for production of essential flavoproteins]. Although nutritional experiments have indicated some (unknown) metabolic or nutritional role for PQQ in mammals, it cannot seriously be accepted as a vitamin until an enzyme can be shown to require it as its cofactor. About one year ago Kasahara and Kato claim to have provided this evidence and announced ‘A new redox-cofactor vitamin for mammals' in Nature. This was greeted with enthusiasm by Reuters news agency “The first new vitamin for 55 years”, and its exploitation by Mitsubishi seems to be underway. However, the claim of Kasahara and Kato was based on sequence analysis of an enzyme, predicted to be involved in mouse lysine metabolism, using databases and search engines which inappropriately label beta propeller sequences as PQQ-binding sites. The ‘sites' wrongly identified by the databases do not represent PQQ-binding sites but represent the beta-sheets that form the ‘blades' of the ‘propeller fold' which happens to be a feature of all PQQ-dependent dehydrogenases, whose main structure is a superbarrel made up of either six or eight ‘propeller blades'. What the evidence actually suggests is that their (predicted) enzyme is an interesting novel protein having an eight-bladed beta propeller structure; but there is no evidence that it is a PQQ-dependent dehydrogenase. There is also no evidence that this protein has any relevance to lysine metabolism.
We argue that the conclusion that the mouse contains a PQQ-dependent dehydrogenase (and hence that PQQ is a vitamin) is an inappropriate conclusion from the evidence presented, and that the enthusiastic welcome with which this ‘new vitamin' was subsequently greeted was therefore misplaced. Our objections are published in Nature [Felton, L. M. & Anthony, C. Role of PQQ as a mammalian enzyme cofactor? Nature doi:10.1038/nature03322 (2005) ], together with further objections by Rucker and colleagues [Rucker, R., Storms, D., Sheets, A., Tchaparian, E. & Fascetti, A. Nature doi:10.1038/nature03323 (2005)] and a response by the original authors [ Takaoki Kasahara , T. & Kato, T. Nature doi:10.1038/nature03324(2005)].
The electron transport chain from methanol dehydrogenase involves two unusual c-type cytochromes; cytochrome cL is the initial electron acceptor and cytochrome cH mediates electron transfer to the oxidase.
Cytochrome cH : This small cytochrome mediates electron flow from cytochrome cLto the oxidase. Cytochrome cH is the electron donor to the oxidase in methylotrophic bacteria. Its amino acid sequence suggests that it is a typical Class I cytochrome c, but some features of the sequence indicated that its structure might be of special interest. The structure of oxidized cytochrome cH has been solved to 2.0 Å resolution by X-ray diffraction. It has the classical tertiary structure of the Class 1 cytochromes c but bears a closer gross resemblance to mitochondrial cytochrome c than to the bacterial cytochrome c2. The left-hand side of the haem cleft is unique; in particular, it is highly hydrophobic, the usual water is absent, and the “conserved” Tyr67 is replaced by tryptophan. A number of features of the structure demonstrate that the usual hydrogen bonding network involving water in the haem channel is not essential and that other mechanisms may exist for modulation of redox potentials in this cytochrome.
Cytochrome cL : The structure of cytochrome cL from Methylobacterium extorquens has been determined by X-ray crystallography to a resolution of 1.6 A ° . This unusually large, acidic cytochrome is the physiological electron acceptor for the quinoprotein methanol dehydrogenase in the periplasm of methylotrophic bacteria. Its amino acid sequence is completely different from that of other cytochromes but its X-ray structure reveals a core that is typical of class I cytochromes c, having a-helices folded into a compact structure enclosing the single haem c prosthetic group and leaving one edge of the haem exposed. The haem is bound through thioether bonds to Cys65 and Cys68, and the fifth ligand to the haem iron is provided by His69. Remarkably, the sixth ligand is provided by His112, and not by Met109, which had been shown to be the sixth ligand in solution.
Cytochrome cL is unusual in having a disulphide bridge that tethers the long C-terminal extension to the body of the structure. The crystal structure reveals that, close to the inner haem propionate, there is tightly bound calcium ion that is likely to be involved in stabilization of the redox potential, and that may be important in the flow of electrons from reduced pyrroloquinoline quinone in methanol dehydrogenase to the haem of cytochrome cL. As predicted, both haem propionates are exposed to solvent, accounting for the unusual influence of pH on the redox potential of this cytochrome.
Professor Anthony's main areas of research are described in more details on his personal website.