AbstractLignin is an aromatic biopolymer that represents the second largest source of organic carbon in the world, after cellulose, and therefore a promising avenue for obtaining valuable chemicals that would otherwise be derived from fossil fuels. Plant cell walls have evolved lignin with functions in protection and water transport, and as such it is inherently recalcitrant to degradation. This results, in part, from the two most abundant of the three monomer streams (G- and S-monolignols) employing O-methylation of hydroxyl groups on the aromatic ring as a barrier to enzymatic ring cleavage. The prevalence of these moieties makes O-demethylation a key step in catabolising lignin, and one that has been the subject of increasing research focus in recent years. This thesis characterises
the structure and function of a cytochrome P450 system, GcoAB, from a plant-biomass degrading Actinomycete, that performs the O-demethylation reaction converting guaiacol to catechol prior to ring cleavage. The system is further demonstrated to be capable of O-demethylating a wide range of aromatics. This gives GcoAB an advantage over known systems, such as VanAB, that have a relatively narrow substrate range, and metabolically expensive tetrahydrofolate dependent systems such as LigM. Crystal structures of GcoA and GcoB show that GcoA employs a series of hydrophobic residues to coordinate guaiacol in the active site, while GcoB presents a novel domain arrangement, and therefore a new
class P450 reductase enzyme. The monooxygenation of guaiacol to catechol requires two sequential electrons, delivered from NADH via the FAD and 2Fe-2S domains of GcoB to the haem iron in the active site of GcoA. Monolignols with small substitutions at the C1 and C6 positions are productively turned over. However, larger substituents here, and at the C4 position, prevent binding and/or catalysis. Crystal structures of GcoA with two such substrates, syringol and vanillin, highlight Phe169 and Thr296 as important for permitting binding of substrates with moieties at the C4 and C6 positions. Engineering GcoA at these residues is demonstrated to facilitate improved accommodation of these substrates in the active site and, at least in the case of syringol, improved catalysis while maintaining activity on guaiacol. This provides a platform from which to develop microbial pathways to funnel monolignols into useful products, where the plasticity of the active site is of particular interest given the heterogeneous products obtained from various methods of lignin depolymerisation.
|Date of Award||17 Dec 2018|
|Supervisor||John McGeehan (Supervisor)|