Navigating the protein landscape around nitroglycerin reductase

• Main Author: Bertels, Michael N. M.
• Published: Cardiff University 2004
• Subjects: 572
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.583401

Nitro-glycerine reductase (NerA) liberates nitrite from glycerol trinitrate (GTN) and enables Agrobacterium radiobacter to utilise GTN and related explosives as sources of nitrogen for growth. The aim of this thesis was to describe the structure / function relationships of this enzyme through an exploration of the protein landscape. nerA was expressed in <italic>Escherichia coli</italic> (<italic>E. coli</italic>) and the resulting activity was characterised. NerA had activity towards GTN, pentaerythritol tetranitrate (PETN), trinitrotoluene (TNT) 1-chloro-2,4-dinitro-benzene (CDNB) but not against ethylene glycol dinitrate (EGDN) or m-dinitro-benzene (DNB). Reductases native to <italic> E. coli</italic> capable of reducing EGDN, GTN, TNT, CDNB, DNB but not PETN were also found. An assay, based on gel filtration followed by a substrate specificity assay, was developed to distinguish NerA from the <italic>E. coli </italic> reductases and a screening method for the detection of activity towards nitro-organic compounds on solid medium was developed. An expression library (30000 colonies) in <italic>E. coli</italic> of random mutants of nerA was screened for activity towards EGDN. Although a number of tolerated and knock-out mutations (for activity towards GTN) were identified in nerA, none of the mutants had altered substrate specificity. The tolerated and knock-out mutations were evaluated using the tertiary structure of Old yellow enzyme, a homologue of NerA. Analyses of a number of random isolates revealed that the ability to liberate nitrite from GTN is widespread and it is not limited to micro-organisms previously exposed to explosives. The statistical parameters involved in the evolutionary cycle that was used to navigate the protein landscape were analysed. It was concluded that mutagenic PCR can generate only 40% of all amino acid exchanges that are possible and that the majority of these exchanges can be achieved only by one particular mutagenic event.