Investigation of the molecular determinants of the substrate- and stereoselectivity of dihydroxyacetone- dependent aldolases and transaldolases

• Applicant: Professor Dr. Georg Sprenger, since 8/2014
• Subject Area: Biochemistry
• Term: from 2011 to 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 201065813

C-C bonding enzymes, such as aldolases and transaldolases, catalyse the formation of a C-C bond with concomitant control of the stereo configuration at the newly formed stereo centre(s). Aldolases with novel stereo- and substrate selectivities are highly desired for application in biocatalysis, e.g. enantiopure synthesis of pharmaceuticals. Our understanding of the mechanism behind the high stereoselectivity of C-C bonding enzymes is still quite limited. In the proposed project, transaldolase B (TalB) and fructose-6-phosphate aldolase (FSA) of Escherichia coli are used as model systems to gain insights into the molecular determinants of their substrate- and stereoselectivities. TalB transfers a dihydroxyacetone moiety from a ketose donor onto an aldehyde acceptor whereas FSA catalyses an aldol addition using dihydroxyacetone (not DHAP!) as donor. The wealth of structural and mechanistic studies on TalB and FSA allows the prediction of the function of active site residues and the effect of Anne Samland substrate- and stereoselectivity of aldolases their replacements. TalB variants exhibiting a novel stereoselectivity (3S,4S) and broadened donor and acceptor specificity will be generated. Furthermore, TalB will be completely converted to a dihydroxyacetone-dependent aldolase. In parallel, FSA will be converted into a transaldolase and the two trajectories will be compared to get insights into the mechanism determining the different reaction types (lyase and transferase) as well as the evolution of FSA from transaldolases. Innovative screening and selection assays will be developed for screening of mutant libraries. The structural model of the active site will be improved by crystallisation of the new variants and determination of the structures of TalB or FSA in complex with substrates or inhibitors.

• DFG Programme: Research Grants
• Ehemalige Antragstellerin: Dr. Anne Samland, until 4/2014