Structural Determination Of Oxidoreductases With Diversified Substrate And Co-enzyme Preference For Gluconobacter Oxydans

Gluconobacter oxydan is one of the most widely used industrial bacterium for the incomplete oxidization of a wide range of sugars, alcohols and polyols into their respective products. Our lab focus on Gluconobacter oxydan for several years and have taken some positive results. However, most of these achievements are basing on wild-type strain and enzymes. Nowadays, enzyme engineering by directed evolution has become the strategy of choice for tailoring the catalytic, biophysical and molecular recognition properties of target proteins. And our lab have accumulated some techniques and methods of structural biology and computational biology. So this thesis focuses on structure-based rational design of G. oxydans oxidoreductases, and expects to build a working platform as a universal approach to engineer any oxidoreductase from G. oxydans with’designed’substrate or co-enzyme preference. This work was performed in three parts as following.First, structural and functional studies of G. oxydans enoate reductase Gox0502Enoate reductase is a large family of flavin mononucleotide (FMN)-containing, NAD(P)H-dependent oxidoreductases, which catalyze C=C bond reduction of α,β-unsaturated ketones and aldehydes in a high substrate and stereo-specific manner. Our lab have identified and characterized two old yellow enzyme homologs from G. oxydans, Gox0502and Gox2684. In this study, we report the crystal structure of Old Yellow Enzyme family protein Gox0502(a.a1-315) in free form at3.3A. Detailed structural analysis revealed the key residues involved in stereo-specific determination of Gox0502, such as Trp66and Trp100. Structure-based computational analysis suggested the bulky side chains of these tryptophan residues may play important roles in product stereo-selectivity. The introduction of Ile or Phe or Tyr mutation significantly reduced the product diastereoselectivity. We hypothesized that less bulky side chains at these critical residues could create additional free space to accommodate intermediates with different conformations. Notably, the introduction of Phe mutation at residue Trp100increased catalytic activity compared to wild-type Gox0502towards a set of substrates tested, which suggests that a less bulky Phe side chain at residue W100F may facilitate product release.Second, structural and functional studies of G. oxydans SDR protein Gox2253Gox2253from G. oxydans belongs to the short-chain dehydrogenases/reductases (SDR) family, and catalyzes the reduction of heptanal, octanal, nonanal and decanal with NADPH. In this study, we report the crystal structure of Gox2253at2.6A resolution, ternary models of Gox2253mutants in complex with NADH/short-chain aldehydes and propose a structural mechanism of substrate selection. Molecular dynamics simulation shows that hydrogen bonds could form between2’-hydroxyl group in the adenosine moiety of NADH and the side chain of Gox2253mutant after arginine at position42is replaced with tyrosine or lysine. Consistent with the molecular dynamics prediction, Gox2253-R42Y/K mutants can use both NADH and NADPH as a coenzyme implies activity.Third, structural and functional studies of G. oxydans ALDH Gox0499The genome sequence of G. oxydans indicated the presence of five genes encoding soluble proteins of the aldehyde dehydrogenase family (Gox0499is one of them). These proteins are speculated to be important for the intracellular metabolism and detoxification of harmful aldehyde derivatives. In this study, we report crystal structures of Gox0499as well as another ALDH Sp2771C262A mutant in complex with NADP+and SSA. Structural comparison of Gox0499with Sp2771, coupled with mutational analysis, demonstrates that Ser157residue in Sp2771and corresponding Pro159residue in Gox0499play critical structural roles in determining NADP+and NAD+preference for Sp2771and Gox0499, respectively, whereas size and distribution of hydrophobic residues along the substrate binding funnel determine substrate selection. Hence, our work has provided insightful structural information into cofactor and substrate selection by ALDH.