Broadening substrate specificity across short-chain dehydrogenase reductases (SDRs)

Short-chain dehydrogenase reductases (SDRs) form a structural motif for enzymes that catalyze a wide variety of reactions1, even if sequence identity often is low. Substrate promiscuity across SDRs so far has been found in a small number of cases, such as between glucose dehydrogenases and imine reductases.2 The current work demonstrates both experimental and computational protein engineering towards broadening substrate specificity across SDRs with hitherto distinct substrate profiles. As an example, we have investigated two classical SDR groups of enzymes: (GDH) glucose 1-dehydrogenase (EC 1.1.1.47), a thermostable protein which catalyzes oxidation of glucose to gluconic acid with NAD(P)H3 and alcohol dehydrogenases (ADHs), which catalyze the reversible reduction of ketones or aldehydes to alcohols, the former often with exquisite stereoselectivity. In this work, we demonstrate not only broadening of substrate specificity of ADHs but, in addition, the interconversion of GDHs and ADHs.4 We have employed site-directed mutagenesis and combinatorial mutagenesis with focused libraries of the active site environment, as well as structure-guided consensus aided by computational modelling to residues affecting the accessibility of the active site.5 We have tracked thermal and process stability of the generated variants via a novel set of tools, especially Differential Scanning Fluorimetry (DSF) with internal aromatic residue standard and SEC-multi-angle laser scattering (SEC-MALLS) with 4 detectors. Lastly, the structural basis for substrate specificity of proteins with GDH and ADH functionality as well as GDH vs ADH functionality will be discussed. References: Kallberg Y, Oppermann U, Jornvall H, Persson B. “Short-chain dehydrogenases/ reductases (SDRs)” Eur J Biochem. Vol. 269(18):4409-17, 2002. Roth S, et al., “Extended Catalytic Scope of a Well-Known Enzyme: Asymmetric Reduction of Iminium Substrates by Glucose Dehydrogenase”, ChemBioChem, 18, 1703-6, 2017. Vazquez-Figueroa E, Chaparro-Riggers J, Bommarius AS. “Development of a Thermostable Glucose Dehydrogenase by a Structure-Guided Consensus Concept”. ChemBioChem, 8: 2295-2301, 2007. Tassoulas L, Bommarius AS, Bommarius BR. “Design of an aromatic thermostable alcohol dehydrogenase from its glucose dehydrogenase parent”, submitted. Gräff M, Buchholz PCF, Stockinger P, Bommarius B, Bommarius AS, Pleiss J. “The Short-chain Dehydrogenase/Reductase Engineering Database (SDRED): a classification and analysis system for a highly diverse enzyme family, Proteins: Structure, Function, and Bioinformatics 1-9, 2019