Catalytic asymmetric Stetter reaction
Final Report Abstract
The main goals of the project P2 were to identify ThDP-dependent enzymes for 1,4-additions and to characterize them concerning enzyme production, substrate and product range, and enantioselectivity. PigD from Serratia marcescens and MenD from E. coli were the starting points for the work on Stetterases. Determination of the substrate range and (stereo)chemical analysis of ligation products of PigD and four sequence-related enzymes were performed. The successfully proven Stetteraseactivity of all five enzymes substantiates a sequence-activity relationship. Moreover, each of the enzymes shows a preference for selected αβ-unsaturated substrates. Selected transformations were performed on a preparative scale. The proposed crystallization and structure determination of one of the Stetterases, however, was not successful despite several different approaches. MenD, which physiologically catalyzes a Stetter-type reaction, was characterized in detail regarding its acceptor substrate range. Methyl vinyl ketone is a common substrate of the Stetterase group of enzymes and MenD. The addition of α-ketoglutarate to acrylates proceeds with good conversion. The reaction with acrylonitrile was kinetically characterized, optimized, and transferred to preparative scale synthesis, work done in collaboration. The ultimate goal of this project was the in vitro and in vivo application of Stetterases to novel transformations. A hypothesis-driven approach was applied to a MenD-related enzyme from E. coli, SucA. Rationally designed variant SucA-H460I led to excellent conversions and enantioselectivities with aromatic substrates. Moreover, we elucidated the reasons for the low optical purity of enzymatically formed acetoin. Mechanistic studies of different enzymes resulted in the identification of a (nonenzymatic) α-hydroxy-β-keto acid rearrangement–decarboxylation of acetolactate derivatives, and a concise strategy for the enzymatic synthesis of highly enantioenriched (R)- and (S)-acetoin (>90% ee). Finally, the in vivo application of MenD variants was realized. Isochorismate is both the physiological substrate of MenD and a precursor of 2,3-trans-CHD. However, the catalytic efficiency (k cat/KM) of MenD for 2,3-trans-CHD was found to be 6 × 103 times lower than for the isochorismate. To modify the substrate scope of MenD, we proposed a model, which subsequently has been proven by the X-ray structural work of Johnston et al., for the binding of isochorismate. To make 2,3-trans-CHD a preferred substrate, the active site was rationally modified in such way that it refuses the natural substrate. The variant Leu478Thr was expressed in E. coli and showed in vivo a higher conversion of 2,3-trans-CHD. Afterwards, high cell-density fermentations were performed for the variants Asn117Arg and Leu478Thr as well as for the double variant Asn117Arg-Leu478Thr resulting in >70-fold improvement towards the desired selectivity. To further increase the chemical diversity, 2,3-trans-CHA was investigated as a substrate of MenD. Based on our docking model, we identified Arg107 as a hot spot for the conversion of 2,3-trans-CHA. Arg107 was mutated to glutamic acid, lysine, and isoleucine. The variants showed up to 23-fold conversion of 2,3-trans-CHA in comparison with wt-MenD, whereas the conversion of 2,3-trans-CHD was lowered to 5-20% relative activity. In summary, in-depth understanding of the mechanism enabled the application of Stetterase enzymes and rationally designed variants thereof towards many different 1,2- and 1,4-additions. Moreover, their biotechnological applications in combination with metabolic engineering has been realized.
Publications
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The Enzymatic Asymmetric Conjugate Umpolung Reaction. Angew. Chem. Int. Ed. 2010, 49: 6600–6603
Dresen C, Richter M, Pohl M, Luedeke S, Müller M
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Recent Developments in Enzymatic Asymmetric C–C Bond Formation, Adv. Synth. Catal. 2012, 354: 3161–3174
Müller M
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TCA Cycle Involved Enzymes SucA and Kgd, as well as Men D: Efficient Biocatalysts for Asymmetric C-C- Bond Formation. Org. Lett. 2013, 15: 452–455
Beigi M, Waltzer S, Fries A, Eggeling L, Sprenger GA, Müller M
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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
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Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes. Appl. Microbiol. Biotechnol. 2014, 98: 9681–9690
Kasparyan E, Richter M, Dresen C, Walter LS, Fuchs G, Leeper FJ, Wacker T, Andrade, SLA, Kolter G, Pohl M, Müller M
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New Stetter reactions catalyzed by thiamine diphosphate dependent MenD from E.coli, J. Biotechnol. 2014, 191: 64–68
Beigi M, Waltzer S, Zarfei M, Müller M
