Discovery of novel geranylgeranyl reductases and characterization of their substrate promiscuity

Corey W Meadows,Brett M Garabedian,Angelique Chanal,Edward E K Baidoo,Raya Abourjeily,Florence Mingardon,Andria V Rodrigues,Veronica T Benites,Taek Soon Lee

doi:10.1186/s13068-018-1342-2

Abstract

BackgroundGeranylgeranyl reductase (GGR) is a flavin-containing redox enzyme that hydrogenates a variety of unactivated polyprenyl substrates, which are further processed mostly for lipid biosynthesis in archaea or chlorophyll biosynthesis in plants. To date, only a few GGR genes have been confirmed to reduce polyprenyl substrates in vitro or in vivo.ResultsIn this work, we aimed to expand the confirmed GGR activity space by searching for novel genes that function under amenable conditions for microbial mesophilic growth in conventional hosts such as Escherichia coli or Saccharomyces cerevisiae. 31 putative GGRs were selected to test for potential reductase activity in vitro on farnesyl pyrophosphate, geranylgeranyl pyrophosphate, farnesol (FOH), and geranylgeraniol (GGOH). We report the discovery of several novel GGRs exhibiting significant activity toward various polyprenyl substrates under mild conditions (i.e., pH 7.4, T = 37 °C), including the discovery of a novel bacterial GGR isolated from Streptomyces coelicolor. In addition, we uncover new mechanistic insights within several GGR variants, including GGR-mediated phosphatase activity toward polyprenyl pyrophosphates and the first demonstration of completely hydrogenated GGOH and FOH substrates.ConclusionThese collective results enhance the potential for metabolic engineers to manufacture a variety of isoprenoid-based biofuels, polymers, and chemical feedstocks in common microbial hosts such as E. coli or S. cerevisiae.

Highlights

Geranylgeranyl reductase (GGR) is a flavin-containing redox enzyme that hydrogenates a variety of unactivated polyprenyl substrates, which are further processed mostly for lipid biosynthesis in archaea or chlorophyll biosynthesis in plants
Very few geranylgeranyl reductase (GGR) have been confirmed as oxidoreductases, and most enzymes having prenyl reductase activity were derived from species that thrive under extremophilic conditions or utilize photosynthesis for energy transduction [25,26,27,28,29,30,31,32]
A few GGRs within this database have been confirmed by other groups to reduce a wide variety of large prenylated substrates, including geranylgeranyl pyrophosphate (GGPP), di-O-geranylgeranylglyceryl phosphate (DGGGP), geranylgeranylchlorophyll, menaquinone, and dolichol [25,26,27,28,29,30,31,32]

Summary

Introduction

Geranylgeranyl reductase (GGR) is a flavin-containing redox enzyme that hydrogenates a variety of unactivated polyprenyl substrates, which are further processed mostly for lipid biosynthesis in archaea or chlorophyll biosynthesis in plants. The two biosynthetic pathways for terpene monomer biosynthesis are the mevalonate and 1-deoxy-d-xylulose 5-phosphate pathways, where pyruvate is converted into either of the C5 terpene building blocks, isopentenyl pyrophosphate or dimethylallyl pyrophosphate [1, 2]. These monomer units are subsequently fused by various. Reducing equivalents are thought to be derived from a NAD(P)H/ferredoxin reductase, in which electron transfer is conducted throughout the protein and modulated by a conserved active site cysteine within the cofactor binding domain, located directly behind the FAD isoalloxazine ring [31]

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