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J. Bacteriol. doi:10.1128/JB.01939-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Cloning, Solubilization, and Characterization of Squalene Synthase from Thermosynechococcus elongatus BP-1

Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112

^* To whom correspondence should be addressed. Email: poulter{at}chem.utah.edu.

	Abstract

Squalene synthase (SQS) is a bifunctional enzyme, which catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to give presqualene diphosphate (PSPP) and the subsequent rearrangement of PSPP to squalene. These reactions constitute the first pathway specific steps in hopane biosynthesis in Bacteria and sterol biosynthesis in Eukarya. The genes encoding SQS were isolated from hopane-producing bacteria Thermosynechococcus elongatus BP-1, Bradyrhizobium japonicum, and Zymomonas mobilis and cloned into an Escherichia coli expression system. The expressed proteins with a His₆-tag were found exclusively in inclusion bodies when no additives were used in the buffer. After extensive optimization, soluble recombinant T. elongatus BP-1 SQS was obtained when cells were disrupted and purified in buffers containing glycerol. The recombinant B. japonicum and Z. mobilis SQS could not be solubilized in any of the expression and purification conditions used. Purified T. elongatus His₆-SQS gave a single band at 42 kDa by SDS-PAGE and molecular ion at m/z 41886 by electrospray mass spectrometry. Incubation with FPP and NADPH gave squalene as the sole product. Incubation of the enzyme with [¹⁴C]FPP in the absence of NADPH gave PSPP. The enzyme requires Mg²⁺ for activity, has an optimum pH of 7.6, and is strongly stimulated by detergent. Under optimal conditions, K_m^FPP = 0.97 ± 0.10 µM and k_cat = 1.74 ± 0.04 s^-1. Zaragozic acid A, a potent inhibitor of mammalian, fungal and yeast SQS, also inhibited recombinant T. elongatus BP-1 SQS, with an IC₅₀ = 95.5 ± 13.6 nM.