Structural Analysis of an Evolved Transketolase Reveals Divergent Binding Modes.

The S385Y/D469T/R520Q variant of E. coli transketolase was evolved previously with three successive smart libraries, each guided by different structural, bioinformatical or computational methods. Substrate-walking progressively shifted the target acceptor substrate from phosphorylated aldehydes, towards a non-phosphorylated polar aldehyde, a non-polar aliphatic aldehyde, and finally a non-polar aromatic aldehyde. Kinetic evaluations on three benzaldehyde derivatives, suggested that their active-site binding was differentially sensitive to the S385Y mutation. Docking into mutants generated in silico from the wild-type crystal structure was not wholly satisfactory, as errors accumulated with successive mutations, and hampered further smart-library designs. Here we report the crystal structure of the S385Y/D469T/R520Q variant, and molecular docking of three substrates. This now supports our original hypothesis that directed-evolution had generated an evolutionary intermediate with divergent binding modes for the three aromatic aldehydes tested. The new active site contained two binding pockets supporting π-π stacking interactions, sterically separated by the D469T mutation. While 3-formylbenzoic acid (3-FBA) preferred one pocket, and 4-FBA the other, the less well-accepted substrate 3-hydroxybenzaldehyde (3-HBA) was caught in limbo with equal preference for the two pockets. This work highlights the value of obtaining crystal structures of evolved enzyme variants, for continued and reliable use of smart library strategies

Engineering Transketolase for Industrial Biotechnology

Transketolase is a ubiquitous enzyme of the thiamine diphosphate-dependent (TPP) family involved in the Calvin cycle and the pentose phosphate pathway. Substrate-walking of E. coli transketolase progressively shifted the target acceptor substrate from phosphorylated aldehydes to non-polar aromatic aldehydes. However, its applicability as an industrial biocatalyst is limited by the lack of combination mutants exhibiting satisfactory substrate breadth and stability. The S385Y/D469T/R520Q variant, which had previously been thought to exhibit differential binding to aromatic substrates, was analysed. Three model substrates were docked into its active site thus revealing two binding pockets supporting π-π stacking interactions. Screening of this variant with other cyclic compounds revealed evolved activities towards valuable industrial building blocks including 4-(methylsulfonyl)benzaldehyde (4-MSBA), a precursor to thiamphenicol. A quadruple mutant was consequently engineered by recombining a stabilising mutation and used as a template for further evolution towards bulky aromatics. Site-directed mutagenesis of a key residue generated the H192P/S385Y/L466M/D469T/R520Q variant which exhibited 5.6-fold improved kinetics towards 4-MSBA compared to the triple mutant. The transition of TK from a model enzyme to a robust industrial biocatalyst however does not only rely on its ability to synthesise novel therapeutic molecules, but also on its thermo- and solvent-stability. 52 variants of TK across the tree of life were consequently aligned to engineer a consensus variant and reconstruct a common ancestor to TK speculated to have branched from proteobacteria, firmicutes and fungi. The resulting common ancestor exhibited trace levels of non-native activity towards non-phosphorylated sugars and provided an initial soluble enzyme to explore the stability/activity relationship of future de novo TKs