A multisubstrate reductase from Plantago major: structure-function in the short chain reductase superfamily

Abstract

The short chain dehydrogenase/reductase superfamily (SDR) is a large family of NAD(P)H-dependent enzymes found in all kingdoms of life. SDRs are particularly well-represented in plants, playing diverse roles in both primary and secondary metabolism. In addition, some plant SDRs are also able to catalyse a reductive cyclisation reaction critical for the biosynthesis of the iridoid backbone that contains a fused 5 and 6-membered ring scaffold. Mining the EST database of Plantago major, a medicinal plant that makes iridoids, we identified a putative 5β-progesterone reductase gene, PmMOR (P. major multisubstrate oxido-reductase), that is 60% identical to the iridoid synthase gene from Catharanthus roseus. The PmMOR protein was recombinantly expressed and its enzymatic activity assayed against three putative substrates, 8-oxogeranial, citral and progesterone. The enzyme demonstrated promiscuous enzymatic activity and was able to not only reduce progesterone and citral, but also to catalyse the reductive cyclisation of 8-oxogeranial. The crystal structures of PmMOR wild type and PmMOR mutants in complex with NADP+ or NAD+ and either 8-oxogeranial, citral or progesterone help to reveal the substrate specificity determinants and catalytic machinery of the protein. Site-directed mutagenesis studies were performed and provide a foundation for understanding the promiscuous activity of the enzyme.

Enhancing Substrate Preference of Iridoid Synthase via Focused Polarity-Steric Mutagenesis Scanning

“…A total of five models were generated, and the top-ranked model was selected for subsequent analysis. Consistent with known ISYs, − the model of Nm ISY2 exhibited a symmetric homodimeric structure. Each dimer comprised two domains, including the dinucleotide-binding Rossman domain and the helical capping domain, and the gap between these two domains constitutes the binding pocket.…”

“…The analysis presented above showed that the tryptophan gating and microenvironment of the substrate tunnel played a crucial role in the substrate preference of Nm ISY2. To investigate the generality of the mechanism, Nc ISY2 from Nepeta cataria , Pm MOR from Plantago major , and Cr ISY were mutated to W107Y/K343M/W345D ( Nc ISY2-3M+), W106Y/V340M/W342D ( Pm MOR-3M+), and W109Y/A339M/W341D ( Cr ISY-3M+), respectively. In the case of Nc ISY2-3M+ and Pm MOR-3M+, the SP value exhibited an increase of 19.2% and 135.5%, respectively, in comparison to their respective wild types.…”

The Progesterone 5β-Reductase/Iridoid Synthase Family: A Catalytic Reservoir for Specialized Metabolism across Land Plants

“…In contrast, progesterone 5β-reductase activity was observed at various degrees in all PRISE homologues, including C. roseus ISY. In PRISEs that accept such linear enones, the molecular flexibility of the substrate, as shown in at least one study on Plantago major PRISE, 32 might render some promiscuity toward other structures. In PRISEs that only accept steroidlike enones, the rigidity of these four-ring structures could affort higher specificity at the expense of activities toward linear enones.…”

Characterization of a putative tropinone reductase from Tarenaya hassleriana with a broad substrate specificity

“…20,21 SDRs were particularly representative in plants. 22 TRs were in the SDR65C group. According to sequence similarity, TRLs were combined in this group.…”

“…The feature‐rich SDRs superfamily forms one of the largest and oldest families of NAD(P)(H)‐dependent oxidoreductases 20,21 . SDRs were particularly representative in plants 22 . TRs were in the SDR65C group.…”