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Biosynthesis of squalene from farnesyl diphosphate in bacteria: three steps catalyzed by three enzymes

Pan JJ, Solbiati JO, Ramamoorthy G, Hillerich BS, Seidel RD, Cronan JE, Almo SC, Poulter CD, (2015) ACS Cent Sci, 1, 77-82. PMCID: Pending

Gene cluster analysis led the IS Bridging Project to investigate an uncharacterized four-gene cassette with a general annotation of "hopanoid biosynthesis". This cassette occurs in genomes spanning a variety of bacterial phyla. Expressing genes in E. coli allowed the Bridging Project to isolate and characterize enzymatic products, while allowing the Microbiology Core to quantify squalene production in vivo. A new biosynthetic pathway, unique to bacteria, is uncovered.


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.

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figure Abstract Image

figureScheme 1: Conversion of FPP to SQ in Two Steps by Eukaryotic SQases

figure Figure 1: Hopanoid biosynthesis clusters in Z. mobilis.

figure Figure 2: Promiscuous products from incubation of yeast SQase with FPP in the absence of NADPH.

figure Figure 3: Radio-TLC activity assays from HpnC-E from (A) Z. mobilis and (B) R. palustris.

figure Scheme 2: Conversion of FPP to SQ in Bacteria: Three Steps/Three Enzymes.

Reprinted with permission from Pan et al. ACS Central Science, doi: 10.1021/acscentsci.5b00115. © 2015 American Chemical Society.