Amrita University
Amritapuri, Vallikavu, Kerala 690525
India
Sathya Srinivasachari and Ramaswamy Subramanian
Biocatalytic Role of Eukaryotic Rieske Oxygenases, DAF-36 and Nvd
Rieske non-heme iron oxygenases (RO) constitute a well-studied class of enzymes in prokaryotes. The oxygenase component, together with a reductase and sometimes a ferredoxin, form a multicomponent RO system. In prokaryotes, ROs activate relatively inert carbon-carbon bonds to initiate the aerobic catabolism of aromatic compounds. They carry out a variety of reactions, such as dihydroxylation, monohydroxylation, desaturation, sulfoxidation, and dealkylation in stereo and regio- specific manner. The versatility of these enzymes makes them useful for large-scale biosynthesis of chiral compounds. Although the structure and function of different prokaryotic ROs have been extensively characterized, similar studies of eukaryotic ROs have not been reported. The structural information of these enzymes can help us to manipulate and modify these enzymes to improve the efficiency as a biocatalyst. Several conserved genes that encode predicted ROs in eukaryotic systems have been identified. Here, we focus on two of these: DAF-36 from C. elegans (nematode worm) and Neverland (Nvd) from D. melanogaster (fruit-fly). DAF-36 and Nvd play a major role in the early stages of steroid hormone biosynthesis, primarily in the conversion of cholesterol of dehydrocholesterol. It is noteworthy that the proposed RO-catalyzed reaction is oxygen-dependent desaturation and monohydroxylation, neither of which is well characterized even in prokaryotes. In our study, we will first determine the structure of these proteins using X-ray crystallography. Following this, we will measure the specific activity and thermodynamic parameters of the ROs (both wildtype and mutated) using steady-state kinetic and isothermal titration calorimetry (ITC) experiments. These experiments will provide information on how and why the active site of the enzymes and the orientation of substrate control the regio- and stereo-selectivity of the products. These studies will help us engineer these enzymes and design biocatalysts to catalyze a variety of chiral organic transformations that can be of relevance to pharmaceutical companies.