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Discovery of novel geranylgeranyl reductases and characterization of their substrate promiscuity.

ABSTRACT: Background: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. To date, only a few GGR genes have been confirmed to reduce polyprenyl substrates in vitro or in vivo. Results:In 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. Conclusion:These 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.

SUBMITTER: Meadows CW

PROVIDER: S-EPMC6309074 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Discovery of novel geranylgeranyl reductases and characterization of their substrate promiscuity.

Meadows Corey W CW Mingardon Florence F Garabedian Brett M BM Baidoo Edward E K EEK Benites Veronica T VT Rodrigues Andria V AV Abourjeily Raya R Chanal Angelique A Lee Taek Soon TS

Biotechnology for biofuels 20181228

<h4>Background</h4>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. To date, only a few GGR genes have been confirmed to reduce polyprenyl substrates in vitro or in vivo.<h4>Results</h4>In this work, we aimed to expand the confirmed GGR activity space by searching for novel genes that function under amenabl ...[more]

PMID: 30607175

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Project description:Archaeal membrane lipids consist of branched, saturated hydrocarbons distinct from those found in bacteria and eukaryotes. Digeranylgeranylglycerophospholipid reductase (DGGR) catalyzes the hydrogenation process that converts unsaturated 2,3-di-O-geranylgeranylglyceryl phosphate to saturated 2,3-di-O-phytanylglyceryl phosphate as a critical step in the biosynthesis of archaeal membrane lipids. The saturation of hydrocarbon chains confers the ability to resist hydrolysis and oxidation and helps archaea withstand extreme conditions. DGGR is a member of the geranylgeranyl reductase family that is also widely distributed in bacteria and plants, where the family members are involved in the biosynthesis of photosynthetic pigments. We have determined the crystal structure of DGGR from the thermophilic heterotrophic archaea Thermoplasma acidophilum at 1.6 Å resolution, in complex with flavin adenine dinucleotide (FAD) and a bacterial lipid. The DGGR structure can be assigned to the well-studied, p-hydroxybenzoate hydroxylase (PHBH) SCOP superfamily of flavoproteins that include many aromatic hydroxylases and other enzymes with diverse functions. In the DGGR complex, FAD adopts the IN conformation (closed) previously observed in other PHBH flavoproteins. DGGR contains a large substrate-binding site that extends across the entire ligand-binding domain. Electron density corresponding to a bacterial lipid was found within this cavity. The cavity consists of a large opening that tapers down to two, narrow, curved tunnels that closely mimic the shape of the preferred substrate. We identified a sequence motif, PxxYxWxFP, that defines a specificity pocket in the enzyme and precisely aligns the double bond of the geranyl group with respect to the FAD cofactor, thus providing a structural basis for the substrate specificity of geranylgeranyl reductases. DGGR is likely to share a common mechanism with other PHBH enzymes in which FAD switches between two conformations that correspond to the reductive and oxidative half cycles. The structure provides evidence that substrate binding likely involves conformational changes, which are coupled to the two conformational states of the FAD.

Dataset Information

Discovery of novel geranylgeranyl reductases and characterization of their substrate promiscuity.

Publications

Discovery of novel geranylgeranyl reductases and characterization of their substrate promiscuity.

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