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

Lead Research Organisation: Queen's University of Belfast
Department Name: Sch of Biological Sciences

Abstract

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.

Technical Summary

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.

Publications

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Description The project was a fundamental study of a process in nature that is important in the recycling of both natural and human made pollutant materials in the environment. A key step the recycling of natural and human made chemicals is reaction with oxygen. This is done by a wide range of oxygenase enzymes in bacteria found widely in the environment. This study specifically looked at a novel group of microbial oxygenases that "fix" molecular oxygen into aromatic compounds. The study focussed on investigating and engineering a novel naphthalene dioxygenase (NDO) electron transfer system from a widely dispersed group of bacteria called Rhodococcus. This would demonstrate its general utility in many bioprocesses. This would also provide background understanding of how dioxygenase enzymes can be used to produce chiral compounds such as cis-dihdrodiols as synthetic pre-cursors in pharmaceutical development. Furthermore, there is a growing use of microorganisms in the clean-up of polluted environments where the precise biochemical mechanisms are not fully understood. The enzymes themselves consist of a complex series of proteins that are a technical challenging to study. We assigned the role of genes (nar genes) involved in functioning of the NDO complex for all of the proteins involved. This involved reductase activity exhibited by the second enzyme in the pathway. A further role for proteins such as Rub1, Rub2 and the NarK was shown by investigating all permutations and a wide range of molar ratios of protein with respect to the NDO protein (or Iron Sulfur Protein (ISP) NarAaAb complex). This in turn showed surprising peroxidase activity that holds out a promise of wider exploitation of NDO in biocatalysis. EPR spectra of the ISP (NarAaAb complex) also indicated the possibility of cycling of electrons between the components of the structure. We observed that the nar genes are conserved in many Rhodococcus strains. However it was surprising that the cluster does not encode reductase that was expected from studies on other bacteria. Instead the acquisition of these genes requires an exogenous (i.e not encoded within the cluster) reductase to mediate activity. These observations greatly inform our understanding of a novel mechanism that it is widespread in nature.
Exploitation Route The impact of the findings has been in two main areas of activity.
Firstly, in studies of environment clean-up technology or bioremediation whereby microorganisms are deployed in polluted soils and ground waters. Secondly, in the exploitation of these enzymes in developing chiral pharmaceutical compounds. Both of these involve direct interaction with industry. These are summarised in the impact narrative.
Sectors Chemicals,Environment,Pharmaceuticals and Medical Biotechnology

 
Description There has been a considerable exchange of results from this project in relation to the environmental remediation activities of the QUESTOR Centre industrial membership (see http://questor.qub.ac.uk/). As a result several projects involving the Centre have benefited from the exchange of data. These include grants awarded for Post-doctoral researchers: 1. Discovery of new bio-molecular analytical approach for assessment of complex microbial processes in bioremediation technologies. Invest Northern Ireland R and D with industrial partner Whiteford Geoservices Ltd (Reg No: NI44990 14 employees, annual turnover ?1.4m). (36 months: 2009-1012: ? 294928). 2. Developing scaled-up biotransformations (Knowledge Transfer Partnership KTP008095 Allen & Boyd 24 months ?124,624 with ALMAC Sciences Ltd). Support for related PhD studentship projects partly supported by the QUESTOR centre: 1. The Regulation and Evolution of Catabolic Gene Networks in the Genus Rhodococcus (Department of Education and Learning -DEL NI: 2008-2011) 2. Exploiting the potential of biological reduction in waste and water treatment systems DEL NI CAST Award and Questor Centre Shell UK and BP Global 2008-2011 3. Directed Evolution Of Oxidoreductases For Biocatalytic Applications (DEL NI: 2008 -2011) 4. Understanding the fate of methylated PAHs in the terrestrial environment and groundwater. (DELNI: 2008 -2011) 5. Investigating Potential Monooxygenation Reactions by Naphthalene Dioxygenase from Rhodococcus sp. NCIMB12038. (DELNI: 2008 -2011) Support for one Early Stage Training (EST) Research Fellow in area of bioremediation as part of: Advanced technologies for Water Resource Management - ATWARM (EC Framework7 Project: Marie Curie Initial Training Networks (ITN):FP7 Project 238273 2009-20013: Euro 3,497,503 Scientist in Charge Professor M Larkin. Collaborators in Germany, Ireland, UK and USA - with overall coordination through QUESTOR) Understanding the molecular science of aromatic waste degrading enzymes. A collaborative grant with Dr Rebecca Pogni, Univertsity of Sienna assisted in the collaboration in carrying out epr specra and discovering the peroxidase activity of the NDO (British-Italian Partnership Programme British Council ?4,000).
First Year Of Impact 2000
Sector Chemicals,Environment,Pharmaceuticals and Medical Biotechnology
Impact Types Economic