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Biosynthesis of Squalene from Farnesyl Diphosphate in Bacteria: Three Steps Catalyzed by Three Enzymes.

ABSTRACT: Squalene (SQ) is an intermediate in the biosynthesis of sterols in eukaryotes and a few bacteria and of hopanoids in bacteria where they promote membrane stability and the formation of lipid rafts in their hosts. The genes for hopanoid biosynthesis are typically located on clusters that consist of four highly conserved genes-hpnC, hpnD, hpnE, and hpnF-for conversion of farnesyl diphosphate (FPP) to hopene or related pentacyclic metabolites. While hpnF is known to encode a squalene cyclase, the functions for hpnC, hpnD, and hpnE are not rigorously established. The hpnC, hpnD, and hpnE genes from Zymomonas mobilis and Rhodopseudomonas palustris were cloned into Escherichia coli, a bacterium that does not contain genes homologous to hpnC, hpnD, and hpnE, and their functions were established in vitro and in vivo. HpnD catalyzes formation of presqualene diphosphate (PSPP) from two molecules of FPP; HpnC converts PSPP to hydroxysqualene (HSQ); and HpnE, a member of the amine oxidoreductase family, reduces HSQ to SQ. Collectively the reactions catalyzed by these three enzymes constitute a new pathway for biosynthesis of SQ in bacteria.

SUBMITTER: Pan JJ

PROVIDER: S-EPMC4527182 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A versatile photoactivatable probe designed to label the diphosphate binding site of farnesyl diphosphate utilizing enzymes.

Project description:Farnesyl diphosphate (FPP) is a substrate for a diverse number of enzymes found in nature. Photoactive analogues of isoprenoid diphosphates containing either benzophenone, diazotrifluoropropionate or azide groups have been useful for studying both the enzymes that synthesize FPP as well as those that employ FPP as a substrate. Here we describe the synthesis and properties of a new class of FPP analogues that links an unmodified farnesyl group to a diphosphate mimic containing a photoactive benzophenone moiety; thus, importantly, these compounds are photoactive FPP analogues that contain no modifications of the isoprenoid portion of the molecule that may interfere with substrate binding in the active site of an FPP utilizing enzyme. Two isomeric compounds containing meta- and para-substituted benzophenones were prepared. These two analogues inhibit Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) with IC(50) values of 5.8 (meta isomer) and 3.0 microM (para isomer); the more potent analogue, the para isomer, was shown to be a competitive inhibitor of ScPFTase with respect to FPP with a K(I) of 0.46 microM. Radiolabeled forms of both analogues selectively labeled the beta-subunit of ScPFTase. The para isomer was also shown to label Escherichia coli farnesyl diphosphate synthase and Drosophila melanogaster farnesyl diphosphate synthase. Finally, the para isomer was shown to be an alternative substrate for a sesquiterpene synthase from Nostoc sp. strain PCC7120, a cyanobacterial source; the compound also labeled the purified enzyme upon photolysis. Taken together, these results using a number of enzymes demonstrate that this new class of probes should be useful for a plethora of studies of FPP-utilizing enzymes.

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Farnesyl Diphosphate (FPP) Toxicity in E. coli, M9 medium

2011-07-07 | GSE30403 | GEO

Farnesyl Diphosphate (FPP) Toxicity in E. coli, LB medium

2011-05-13 | GSE29267 | GEO

Synthesis of frame-shifted farnesyl diphosphate analogs.

Project description:A set of synthetic approaches were developed and applied to the synthesis of eight frame-shifted farnesyl diphosphate (FPP) analogs. These analogs bear increased or decreased methylene units between the double bonds and/or diphosphate moieties of the isoprenoid structure. Evaluation versus mammalian FTase revealed that small structural changes can lead to dramatic changes in substrate ability.

| S-EPMC6863160 | biostudies-literature

Farnesyl Diphosphate (FPP) Toxicity in E. coli, LB medium

Project description:This experiment studied the effect of FPP accumulation on E. coli. E. coli cells transformed with pMBIS (the S. cerevisiae mevalonate pathway enzymes converting mevalonate to FPP) and fed mevalonate produce large amounts of FPP, which causes toxicity when it accumulates. When coupled with an active amorphadiene synthase (pADS) the cells produce amorphadiene, a non-toxic isoprenoid. To accumulate FPP, but maintain similar protein burden, an amorphadiene synthase with 3 mutations to render it inactive was used (pADSmut) to accumulate FPP. E. coli was transformed with pMBIS and pADS or pMBIS and pADSMut and grown in LB and fed 10 mM mevalonate and induced with 0.5 mM IPTG, then sampled at subsequent time points. This comparison is between E. coli DH1 cells accumulating FPP via the heterologous mevalonate pathway (pMBIS/pADSmut) to cells producing amorphadiene via the same pathway (pMBIS/pADS). Samples were collected 2.5, 5, 7, and 29.5 hr after addition of IPTG and mevalonate. One biological replicate was used. Total RNA was extracted, reverse transcribed, labeled, and hybridized to multiple slides for technical replicates.

2011-05-13 | E-GEOD-29267 | biostudies-arrayexpress

Farnesyl Diphosphate (FPP) Toxicity in E. coli, M9 medium

Project description:This experiment studied the effect of FPP accumulation on E. coli. E. coli cells transformed with pMBIS (the S. cerevisiae mevalonate pathway enzymes converting mevalonate to FPP) and fed mevalonate produce large amounts of FPP, which causes toxicity when it accumulates. When coupled with an active amorphadiene synthase (pADS) the cells produce amorphadiene, a non-toxic isoprenoid. To accumulate FPP, but maintain similar protein burden, an amorphadiene synthase with 3 mutations to render it inactive was used (pADSmut) to accumulate FPP. E. coli was transformed with pMBIS and pADS or pMBIS and pADSMut and grown in M9+glucose with varying magnesium concentrations and fed 20 mM mevalonate and induced with 0.5 mM IPTG, then sampled at subsequent time points. This comparison is between E. coli DH1 cells accumulating FPP via the heterologous mevalonate pathway (pMBIS/pADSmut) to cells producing amorphadiene via the same pathway (pMBIS/pADS). Samples were collected 6, 10, 14, and 26.5 hr after addition of IPTG and mevalonate. One biological replicate was used. Total RNA was extracted, reverse transcribed, labeled, and hybridized to multiple slides for technical replicates.

2011-07-07 | E-GEOD-30403 | biostudies-arrayexpress

A cytosolic bifunctional geranyl/farnesyl diphosphate synthase provides MVA-derived GPP for geraniol biosynthesis in rose flowers.

Project description:Geraniol derived from essential oils of various plant species is widely used in the cosmetic and perfume industries. It is also an essential trait of the pleasant smell of rose flowers. In contrast to other monoterpenes which are produced in plastids via the methyl erythritol phosphate pathway, geraniol biosynthesis in roses relies on cytosolic NUDX1 hydrolase which dephosphorylates geranyl diphosphate (GPP). However, the metabolic origin of cytosolic GPP remains unknown. By feeding Rosa chinensis "Old Blush" flowers with pathway-specific precursors and inhibitors, combined with metabolic profiling and functional characterization of enzymes in vitro and in planta, we show that geraniol is synthesized through the cytosolic mevalonate (MVA) pathway by a bifunctional geranyl/farnesyl diphosphate synthase, RcG/FPPS1, producing both GPP and farnesyl diphosphate (FPP). The downregulation and overexpression of RcG/FPPS1 in rose petals affected not only geraniol and germacrene D emissions but also dihydro-β-ionol, the latter due to metabolic cross talk of RcG/FPPS1-dependent isoprenoid intermediates trafficking from the cytosol to plastids. Phylogenetic analysis together with functional characterization of G/FPPS orthologs revealed that the G/FPPS activity is conserved among Rosaceae species. Site-directed mutagenesis and molecular dynamic simulations enabled to identify two conserved amino acids that evolved from ancestral FPPSs and contribute to GPP/FPP product specificity. Overall, this study elucidates the origin of the cytosolic GPP for NUDX1-dependent geraniol production, provides insights into the emergence of the RcG/FPPS1 GPPS activity from the ancestral FPPSs, and shows that RcG/FPPS1 plays a key role in the biosynthesis of volatile terpenoid compounds in rose flowers.

| S-EPMC10175749 | biostudies-literature

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