Cloning, solubilization, and characterization of squalene synthase from Thermosynechococcus elongatus BP-1.
Squalene synthase (SQS) is a bifunctional enzyme that 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 the 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 His(6) 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 SQSs could not be solubilized under any of the expression and purification conditions used. Purified T. elongatus His(6)-SQS gave a single band at 42 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis 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 [(14)C]FPP in the absence of NADPH gave PSPP. The enzyme requires Mg(2+) for activity, has an optimum pH of 7.6, and is strongly stimulated by detergent. Under optimal conditions, the K(m) of FPP is 0.97 +/- 0.10 microM and the k(cat) is 1.74 +/- 0.04 s(-1). Zaragozic acid A, a potent inhibitor of mammalian, fungal, and Saccharomyces cerevisiae SQSs, also inhibited recombinant T. elongatus BP-1 SQS, with a 50% inhibitory concentration of 95.5 +/- 13.6 nM.
Lee
Sungwon
S
Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112, USA.
Poulter
C Dale
CD
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D015636:Magnesium Chloride; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011136:Polysorbates; D016415:Sequence Alignment; D012995:Solubility; D013185:Squalene / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
D011136:Polysorbates; D015636:Magnesium Chloride; D013185:Squalene; D013186:Farnesyl-Diphosphate Farnesyltransferase