Identifying structural features for changing substrate preferences in two Plantago PRISEs (pro-gesterone 5 beta-reductase and / or iridoid synthase)

Dorfner M, Klein J, Lanig H, Kreis W, Munkert J (2021)

Publication Type: Conference contribution

Publication year: 2021

Journal

Publisher: GEORG THIEME VERLAG KG

City/Town: STUTTGART

Pages Range: 1303-1303

Conference Proceedings Title: PLANTA MEDICA

DOI: 10.1055/s-0041-1736952

Abstract

Cardenolides are drugs used to treat congestive heart failure. Recently their antiproliferative action was brought back into focus [1]. They are still extracted from plants, as their chemical structure impedes chemical synthesis. Though attempts have been made to produce cardenolides by plant tissue culture, farming foxglove still remains the sole source of cardenolides with a possible alternative approach based on microbial production systems. So far we engineered yeast and E. coli to produce intermediate products of cardenolide biosynthesis but still need to overcome enzyme bottlenecks [2]. Targets of enzyme engineering are the PRISEs (progesterone-5β-reductase/iridoid synthase-like enzymes), that catalyzes the reduction of progesterone to 5β-pregnane-3,20-dione, which is an important intermediate of cardenolide biosynthesis. PRISEs occur ubiquitously in the plant kingdom [3] and are enzymes with relaxed substrate specificity [4].

In biotechnology shaping enzymes with respect to their substrate specificity is a useful approach to optimize biosynthetic pathways. Thus, to overcome substrate shortages, we investigated substrate conversion in silico and in vitro of two Plantago PRISEs by creating mutants via site-directed mutagenesis. A PRISE of P. media showed a 70-fold higher specific activity with progesterone than its orthologue from P. lanceolata. Docking substrates into mutants prior to the site-directed mutagenesis, was used as additional tool to identify catalytically impactful residues, so that we were able to completely invert the substrate specificities of the two Plantago PRISEs for progesterone. Engineering substrate specificity into promiscuous enzymes may not only help to eliminate side reactions in biosynthetic pathway but also to understand metabolite-enzyme evolution

Authors with CRIS profile

Involved external institutions

Friedrich-Schiller-Universität Jena Germany (DE)

How to cite

APA:

Dorfner, M., Klein, J., Lanig, H., Kreis, W., & Munkert, J. (2021). Identifying structural features for changing substrate preferences in two Plantago PRISEs (pro-gesterone 5 beta-reductase and / or iridoid synthase). In PLANTA MEDICA (pp. 1303-1303). STUTTGART: GEORG THIEME VERLAG KG.

MLA:

Dorfner, Maja, et al. "Identifying structural features for changing substrate preferences in two Plantago PRISEs (pro-gesterone 5 beta-reductase and / or iridoid synthase)." Proceedings of the PLANTA MEDICA STUTTGART: GEORG THIEME VERLAG KG, 2021. 1303-1303.

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