Development of a novel electron transfer system for oxido-reduction bioprocesses using rhodococci

The fixation of molecular oxygen into organic compounds is one of the key steps in the biodegradative side of the carbon and oxygen cycle in nature in that it initiates the aerobic degradation of many compounds. This process recycles carbon and is mediated by microbial enzymes called oxygenases. Many aromatic hydrocarbons / such as benzene and naphthalene / are priority pollutants in the environment and their remediation is mediated by such microbial enzymes. In addition, these enzymes have turned out to be very useful in producing high value compounds that are of potential value in the pharmaceutical and chemical industry. Such chemicals are extremely difficult to make chemically. This because the enzyme, unlike cruder chemical reactions, can accurately add chemical groups, in the case of di-oxygenases hydroxyl groups, to produce chiral molecules / that is either one of a pair of compounds that are mirror images on each other but are not identical. The current understanding is that microbial dioxygenases / that incorporate two oxygen atoms into aromatic compounds / employ an electron transfer process from cellular cofactors through a complex of proteins. Recent work here has indicated that we have discovered a very novel and potentially more efficient electron transfer system in soil bacteria called rhodococci. We intend to study the mechanism involved in this process in detail. Exploitation of this oxygenase system in these bacteria has significant implications for the efficient production of new chiral compounds.

We will test three hypotheses regarding Rhodococcus naphthalene dioxygenase function; a) that electrons are recycled from dehydrogenase action directly to the dioxygenase via a novel protein, NarK; b) that NAD and NADH are rapidly recycled after the reaction is 'kick-started'; c) that NarB can act as a reductase in the absence of NarK. This will involve series of experiments; cloning and purification of the postulated components identified in preliminary studies; determination of their structures; analysis of the effects of knock-out and site-directed mutants; determination of all the components and cofactors involved; demonstration of protein-protein interactions; determination of Redox characteristics and balance points for each component and likely reduction sequence and electron flow between them. Some of the proposed work will be in collaboration with Professor Pogni, University of Sienna (EPR spectral determinations) and Professor Ramaswamy, University of Iowa (crystallography). We will also perform as series of biotransformation experiments aiming to develop the Rhodococcus NDO system as a biocatalyst and determine its efficiency with regard to electron transfer. Because the Rhodococcus NDO appears to be able to channel electrons directly from an alcohol (naphthalene cis-diol) to a dioxygenase, it may operate in an extracellular. Ultimately, if we understand the structural and mechanistic characteristics of these proteins that allow them to function we could engineer redox systems that utilise a cheap electron source (such as ethanol) and directly channel electrons to any redox biocatalysts. This goal would completely transform the field of redox biocatalysis. However, there are many steps that need to be take before this can be achieved. The first of these / covered by this proposal / is to understand at the fundamental level the mechanisms employed in the Rhodococcus spp. NCIMB 12038 to facilitate this direct electron transfer property.