Affiliation:
1. Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112
Abstract
ABSTRACT
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 μM 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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
58 articles.
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