title: Second generation engineering of transketolase for polar aromatic aldehyde substrates.
abstract: Transketolase has significant industrial potential for the asymmetric synthesis of carboncarbon bonds with new chiral centres. Variants evolved on propanal were found previously with nascent activity on polar aromatic aldehydes 3-formylbenzoic acid (3-FBA), 4-formylbenzoic acid (4-FBA), and 3-hydroxybenzaldehyde (3-HBA), suggesting a potential novel route to analogues of chloramphenicol. Here we evolved improved transketolase activities towards aromatic aldehydes, by saturation mutagenesis of two active-site residues (R358 and S385), predicted to interact with the aromatic substituents. S385 variants selectively controlled the aromatic substrate preference, with up to 13-fold enhanced activities, and KM values comparable to those of natural substrates with wild-type transketolase. S385E even completely removed the substrate inhibition for 3-FBA, observed in all previous variants. The mechanisms of catalytic improvement were both mutation type and substrate dependent. S385E improved 3-FBA activity via kcat, but reduced 4-FBA activity via KM. Conversely, S385Y/T improved 3-FBA activity via KM and 4-FBA activity via kcat. This suggested that both substrate proximity and active-site orientation are very sensitive to mutation. Comparison of all variant activities on each substrate indicated different binding modes for the three aromatic substrates, supported by computational docking. This highlights a potential divergence in the evolution of different substrate specificities, with implications for enzyme engineering.
authors.affiliation: Department of Biochemical Engineering, University College London, Gordon Street, London WC1H 0AH, UK.
authors.forename: Panwajee
authors.initials: P
authors.lastname: Payongsri
authors.affiliation: Department of Chemistry, University College London, 20 Gordon Street, London WC1H 0AJ, UK.
authors.forename: David
authors.initials: D
authors.lastname: Steadman
authors.affiliation: Department of Chemistry, University College London, 20 Gordon Street, London WC1H 0AJ, UK.
authors.forename: Helen C
authors.initials: HC
authors.lastname: Hailes
authors.affiliation: Department of Biochemical Engineering, University College London, Gordon Street, London WC1H 0AH, UK. Electronic address: p.dalby@ucl.ac.uk.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Dalby
mesh_terms: D000447:Aldehydes / Q000378:metabolism; D019943:Amino Acid Substitution; D020134:Catalytic Domain / Q000235:genetics; D019020:Directed Molecular Evolution; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006841:Hydrocarbons, Aromatic / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014174:Transketolase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000447:Aldehydes; D029968:Escherichia coli Proteins; D006841:Hydrocarbons, Aromatic; D011994:Recombinant Proteins; D014174:Transketolase
keywords: Benzaldehyde; Biocatalysis; Directed evolution; Enzyme engineering; Transketolase