MenD (SEPHCHC synthase), a thiamine-dependent enzyme with similarity to the Stetterases
Zusammenfassung der Projektergebnisse
The main goals of project P4 were a better understanding of structure-function relationships and the substrate spectrum of the ThDP-dependent enzyme, MenD which catalyzes a Stetter-type reaction with its native donor, 2-oxoglutarate, and performs 1,4-additions on αβ-unsaturated acceptor compounds such as isochorismate, acrylic acid, and 2,3-trans-CHD with good conversions. MenD also performs carboligations (1,2-additions) with various aromatic and aliphatic aldehydes as acceptors. 2-oxoacids such as pyruvate, 2-oxobutyrate, oxaloacetate and 2-oxoisovalerate were found to serve as donors for MenD at low or almost negligible rates. In contrast, we found that both enantiomers of the diacid, 4-hydroxy-2-oxoglutarate (HOG), are utilized by MenD for 1,2- and 1,4-additions. This now enables access to highly functionalized products which all display an α-hydroxycarboxylate moiety. Access to the S- and R-HOG donors was made possible by pyruvate-dependent aldolases Eda and DgoA from E. coli, respectively, as C-C ligation catalysts from the simple educts, pyruvate and glyoxylate. Purified S- and R-HOG samples from Eda and DgoA gave clear CD signals and were of opposite kind. The stereoconfigurations of the MenD-catalyzed reactions with these donors are preliminary as the absolute configurations could not be determined. The MenD variant I474A F475G (from project P2) was successfully utilized for 1,4-additions with 2-oxoglutarate and acrylic acid with complete conversion. The variant also performed 1,2-additions with both HOG-enantiomers as donors with benzaldehyde and hexanal, although with significantly lower conversions than by MenD wild type enzyme. Interestingly, however, the I474A F475G variant did not use 2,3-trans-CHD as Michael acceptor, neither with 2-oxoglutarate nor with both HOG enantiomers. This points to an incompatibility of the two substitutions with 2,3-trans-CHD as acceptor and warrants further investigations in the future. In cooperation with the group of Gunter Schneider at Stockholm, crystals of three MenD variants (S32D, R107K, and R413A) were obtained. Apart from the different electron densities for the amino acid substitutions, no major conformational changes were observed. Attempts to crystallize MenD proteins in the presence of the donor compounds 2-oxoglutarate and S-HOG were unsuccessful. Thus, no further insights into structure-function relationships of MenD could be gained. In summary, the substrate spectrum of MenD is larger than known before the start of the project P4. Especially HOG turned out to be an interesting novel and promising donor.
Projektbezogene Publikationen (Auswahl)
- Diversity-Oriented Production of Metabolites Derived from Chorismate and Their Use in Organic Synthesis. Angew. Chem. Int. Ed. 2011, 50: 7781–7786
Bongaerts J, Esser S, Lorbach V, Al-Momani L, Müller MA, Franke D, Grondal C, Kurutsch A, Bujnicki R, Takors R, Raeven L, Wubbolts M, Bovenberg R, Nieger M, Schürmann M, Trachtmann N, Kozak S, Sprenger GA, Müller M
(Siehe online unter https://doi.org/10.1002/anie.201103261) - C–C bond formation using ThDP-dependent lyases. Curr. Opin. Chem. Biol. 2013, 17: 261–270
Müller M, Sprenger GA, Pohl M
(Siehe online unter https://doi.org/10.1016/j.cbpa.2013.02.017) - TCA Cycle Involved Enzymes SucA and Kgd, as well as MenD: 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
(Siehe online unter https://doi.org/10.1021/ol3031186) - Extended substrate range of thiamine diphosphate-dependent MenD enzyme by coupling of two C–C-bonding reactions, Appl. Microbiol. Biotechnol. 2018, 102: 8359–8372
Schapfl M, Baier S, Fries A, Ferlaino S, Waltzer S, Müller M, Sprenger GA
(Siehe online unter https://doi.org/10.1007/s00253-018-9259-z) - ChemBioChem 2019, 20: 1672–1677
Fries A, Mazzaferro LS, Bisel P, Grüning B, Stibal K, Sprenger GA, Buchholz PCF, Pleiss J, Müller M
(Siehe online unter https://doi.org/10.1002/cbic.201900050)
