Structural and functional studies on novel oxidoreductases specific to steroids and isoprenoids reduction

Abstract

Oxidoreductases constitute a large class of enzymes, present in all three domains of life. They use a variety of electron acceptors and electron-donating substrates, yielding many products of pharmacological and biotechnological interest. Aberrations in their function are implicated in various diseases and posing as drug targets. We employed X-ray crystallography and Cryo- electron microscopy techniques for structural and functional investigation of three atypical NADPH-dependent oxidoreductase systems which possess non-canonical oxidoreductase mechanism. Two of them govern the iridoid biosynthesis, a precursor for vincristine and vinblastine (iridoid synthase and 10-hydroxygeraniol dehydrogenase) and the third one, WW domain containing oxidoreductases, is a newly identified drug target for breast cancer. Iridoid synthases (IS), a short-chain dehydrogenase/reductase, involved in the biosynthesis of iridoids, exhibit stringent substrate specificity. Unlike the canonical terpene synthases, IS catalyzes the NADPH-dependent reduction of 10-oxogeranial resulting in the formation of an enol or enolate intermediate, which further cyclizes via Michael addition to yield napetalactol. To understand the structural basis of the catalytic mechanism and substrate specificity of IS we determined the crystal structure of IS. From structural, biochemical and molecular dynamics studies we show that a complex interplay of interactions between the loops at the substrate entry confers the substrate specificity to the enzyme. Furthermore, to understand the stereo selectivity production of napetalactol in a concerted chemical reaction of IS and its precursors enzyme in the pathway, 10-HGO, we probed the structural basis of their interaction using single particle cryo-EM and other biophysical studies. Here we show that 10-HGO and IS indeed interact physically to form a channel between the molecules to facilitate passage of former’s (10-HGO) product as a substrate to the later (IS) in the “handshake” manner. Additionally, the crystal structure of 10-HGO provided insight into its catalytic mechanism of the enzyme. Continuing in the same direction, we addressed the structural basis of catalytic mechanism of a recently identified SDR, WW domain-containing oxidoreductase (WWOX), shown to be implicated in several human cancers. Our studies unravel putative substrates of WWOX, its specificity. We have further successfully crystalized this protein to elucidate the mechanism of the protein. The outcome of these studies has significant implications in biotechnology of alkaloid synthesis and development of novel drugs targeting WWOX.