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.
Highlights
The SDR superfamily has been identified in organisms from viruses to higher eukaryotes, constituting an ancient lineage retained in all kingdoms of life[1]
Comparing the results for PmMOR with the PRISE family members, C. roseus iridoid synthase (CrIS) and Digitalis lanata 5ß-progesterone oxido-reductases (5ß–POR) (Dl5ß–POR), offers new insight into the substrate specificity determinants and catalytic activity of the PRISE enzyme family and suggests that these enzymes are able to act on diverse substrates and perform both reduction and reductive cyclisations
A complete genome of P. major is not yet available, an EST database is accessible and a gene annotated as a 5ß–POR was identified (ADG56541.1) with an E value of 3e−164 based on sequence identity to CrIS, with no other genes with significant homology in the search results[22]. This is consistent with the high degree of sequence similarity between CrIS and 5ß–PORs from other plants[4]
Summary
The SDR superfamily has been identified in organisms from viruses to higher eukaryotes, constituting an ancient lineage retained in all kingdoms of life[1]. Progesterone reductases and iridoid synthases share a high degree of sequence and structural homology, but perform distinct reactions and act in different biosynthetic pathways[4]. In order to investigate the function of this protein, which we termed PmMOR (Plantago major multisubstrate oxido-reductase), we expressed, purified and determined its activity with different substrates including 8-oxogeranial, citral and 5β-progesterone. Comparing the results for PmMOR with the PRISE family members, C. roseus iridoid synthase (CrIS) and Digitalis lanata 5ß–POR (Dl5ß–POR), offers new insight into the substrate specificity determinants and catalytic activity of the PRISE enzyme family and suggests that these enzymes are able to act on diverse substrates and perform both reduction and reductive cyclisations
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