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Engineering of the PETNR active site to accommodate novel α/β substituted enone substrates.
[Thesis]. Manchester, UK: The University of Manchester; 2010.
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Abstract
Experiments facilitating the engineering of the PETNR active site to accommodate a range of non natural enone substrates with substituents localised on the α and β carbons of the unsaturated bond are described. In order to facilitate the high throughput purification of PETNR libraries poly histidine (PETNRHis) and biotin (PETNRBio) tagged PETNR variants were generated. High throughput protocols were developed for the automated generation, purification and screening of libraries in a 96 well format. Protocols were optimised and trialled using blocks consisting of PETNRHis WT only and characterised in terms of intra block variation. A range of single site saturation mutagenic libraries were generated at positions in the active site consisting of T26, Y68, W102, H181, H184, Y186, Q241 and Y351. Sequencing results indicated randomised libraries with the occasional instance of bias evident. Expression and purification in a 96 well format was monitored by SDS PAGE and protein quantitation. Library activity was quantified and demonstrated to retain varying degrees of activity with the model substrate 2-cyclohexenone. Following this verification of the experimental protocol libraries were screened against a range of substrates analogous to substrates demonstrated to be active with PETNRWT but incorporating substituents at the α and β carbons. ‘Hits’ generated from these screening reactions were studied further by the determination of the specific activity and quantitation of substrate / product from biotransformation reactions. From these screening experiments totalling 3,600 individual reactions, 35 were identified as potential hits, of these 8 proved to be genuinely improved variants. Substituents at the β carbon were demonstrated to compromise the activity of the WT enzyme most severely. Positions 68, 102, and 351 were demonstrated to play an important role in the accommodation of substituents at the α carbon whilst residues 26 and 351 are important for the β carbon. The best variants demonstrated up to 9 fold improvements in poor substrates which represented rates in excess of those observed for model substrates.