Catalytic promiscuity of thiamine enzymes

• Antragstellerinnen: Professorin Dr. Martina Pohl; Professorin Dr. Dörte Rother
• Fachliche Zuordnung: Biologische und Biomimetische Chemie ; Biochemie
• Förderung: Förderung von 2010 bis 2018
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 128900243

The project aims on a detailed understanding of factors controlling the stereo- and chemoselectivity of thiamine diphosphate (ThDP)-dependent enzymes. These enzymes catalyze the benzoin condensation of aldehydes to chiral 2-hydroxy ketones. Based on our previous studies on 2-ketoacid decarboxylases we could deduce mainly steric factors influencing the chemo- and stereoselectivity of these enzymes. The current mechanistic model suggests that the arrangement of aldehydes in the active site prior to carboligation is decisive for stereoselectivity. In our model the first aldehyde is bound to the cofactor ThDP (donor) and the second {acceptor) aldehyde is having different options to attack the donor. All wild-type 2-ketoacid decarboxylases and benzaldehyde lyase favour a parallel arrangement of the aldehyde site chains in the active site, which explains their general /^-selectivity. Only few of them, with a special structural element, the S-pocket, allow an anti-parallel approach giving rise to S-products. The development of appropriate variants to increase the range of S-products by a combined approach of structural investigation, modeling and site-directed mutagenesis was successful. Besides, indications for an alternative S-path were found. Based on these results we plan to refine our mechanistic model concerning the pnnciples directing selectivity in ThDPdependent enzymes by a combined approach of structural modeling and enzyme engineenng. One goal is to address currently unsolved synthetic problems, e.g. the synthesis of (S)-benzoins and the mixed carboligation of aromatic and branched-chain aliphatic aldehydes. The second goal is the further refinement of our structural model and the extension of these studies on further enzymes of the decarboxylase (DC)-like family of ThDP-dependent enzymes: Acetohydroxyacid synthase and MenD. In a second project phase we will include transketolase and 1-deoxy-D-xylulose 5-phosphate synthase into our studies which belong to a different structural family of ThDP-dependent enzymes and accept multifunctionalized chiral substrates.

• DFG-Verfahren: Forschungsgruppen
• Teilprojekt zu: FOR 1296: Diversity of Asymmetric Thiamine Catalysis