Catalytic promiscuity of thiamine enzymes
Biochemistry
Final Report Abstract
A model to understand and predict the stereoselectivity of ThDP-dependent decarboxylases, which was initially based on two enzymes (PpBFD and PfBAL) was refined and probed with five other members of the decarboxylase family (EcAHAS, EcMenD, BsMenD, ApPDC and PpYerE). Besides opening of an S-pocket, which is mandatory to switch the stereoselectivity of these R-selective enzymes for the carboligation reaction, the resulting S-pocket should be optimized for the acceptor substrate side chain to achieve optimal stabilization in the pocket. If this approach is not sufficient to achieve high S-selectivity for the target reaction, the original R-pathway of the wildtype enzyme should be blocked. Using this concept, highly S-selective variants for EcMenD, BsMenD, and ApPDC could be obtained. The work on MenDs was specifically mentioned in the “hot of the press” article in Natural Products Reports. Robert Westphal was awarded with two poster prices at the „Trends in Enzymology 2012“ and “Biocat 2012”. Besides, for EcAHAS and PpYerE, which are structurally highly similar, the potential S-pocket is rather small, since an alpha-helix occupies this area to a great extent. In both cases we were not able to design S-selective variants. This is a similar situation observed with PfBAL, where no such pocket is available at al. However, based on the mechanistic model, we were able to design a hybride enzyme making use of the big S-pocket in ApPDC and the large donor binding site in PfBAL for the synthesis of (S)-benzoins. Whereas our simple two-state model can explain the stereoselectivity of decarboxylases with high reliability, it could not immediately be applied to the transketolase family. By expansion of the model to four states the different active site architecture could be taken into account. However, since two binding states, yield the R- and S-product, respectively, it cannot be clearly decided, which binding state is predominantly populated before productive C-C-bond formation. The most challenging work package was WP 2.2 (sequence morphing). Biochemical fingerprinting of selected (potential and not yet characterized) enzyme sequencess from the TEED identified new enzymes with a different reaction spectrum relative to known enzymes from the MenD, BAL and AHAS family. The designed new S-selective enzymes are valuable new biocatalysts for the application in preparative biocatalysis. This was impressively demonstrated by application of the ApPDC triple variant for the production of (S)-PAC in a two-step cascade towards nor(pseudo)ephedrines. At a 4 L scale product concentrations up to 450 mM where obtained with high purity (ee/de > 98%) meeting industrial demands. Small industrial scale preparations are currently in progress. Further, a market potential analysis for further pharmaceutically relevant amino alcohols and tetrahydroisoquinolines is currently under investigation by Enzymicals.
Publications
- Conversion of Pyruvate Decarboxylase into an Enantioselective Carboligase with Biosynthetic Potential. J. Am. Chem. Soc. 2011, 133: 3609–3616
Meyer D, Walter L, Kolter G, Pohl M, Müller M, Tittmann K
(See online at https://doi.org/10.1021/ja110236w) - S-Selective Mixed Carboligation by Structure-Based Design of the Pyruvate Decarboxylase from Acetobacter pasteurianus. ChemCatChem. 2011, 3: 1587–1596
Rother D, Kolter G, Gerhards T, Berthold CL, Gauchenova E, Knoll M, Pleiss J, Müller M, Schneider G, Pohl M
(See online at https://doi.org/10.1002/cctc.201100054) - 2013. (S)-Selective MenD variants from Escherichia coli provide access to new functionalized chiral α-hydroxy ketones. Chem. Commun. 49, 2061–2063
Westphal, R., Waltzer, S., Mackfeld, U., Widmann, M., Pleiss, J., Beigi, M., Müller, M., Rother, D., Pohl, M.
(See online at https://doi.org/10.1039/c3cc38607d) - Tailoring (S)-selectivity of MenD from Escherichia coli. ChemCatChem 2013, 13: 3587–3594
Westphal R, Hahn D, Mackfeld U, Waltzer S, Beigi M, Widmann M, Vogel C, Pleiss J, Müller M, Rother D, Pohl M
(See online at https://doi.org/10.1002/cctc.201300318) - A Tailor-Made Chimeric Thiamine Diphosphate Dependent Enzyme for the Direct Asymmetric Synthesis of (S)-Benzoins. Angew. Chem. Int. Ed. 2014, 53: 9376–9379
Westphal R, Vogel C, Schmitz C, Pleiss J, Müller M, Pohl M, Rother D
(See online at https://doi.org/10.1002/anie.201405069) - Asymmetric synthesis of (S)-phenylacetylcarbinol – closing a gap in C–C bond formation. Green Chem. 2017, 19: 380–384
Sehl T, Bock S, Marx L, Maugeri Z, Walter L, Westphal R, Vogel C, Menyes U, Erhardt M, Müller M, Pohl M, Rother D
(See online at https://doi.org/10.1039/c6gc01803c) - Verfahren zur Herstellung von Cathin durch Kopplung einer (S)- selektiven Lyase und einer (S)-selektiven Transaminase. PCT/EP 3008198, granted: 10.05.2017
Sehl T, Baraibar A G, Pohl M, Rother D
