Affiliation:
1. Pharmaceutical Research Preclinical Infectious Diseases, F. Hoffmann- La Roche Ltd., CH-4070 Basel, Switzerland
Abstract
ABSTRACT
The prenyltransferase undecaprenyl pyrophosphate synthetase (di-
trans
,poly-
cis
-decaprenylcistransferase; EC
2.5.1.31
) was purified from the soluble fraction of
Escherichia coli
by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (
E
,
E
)-[1-
3
H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphos-phate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all
E. coli
proteins yielded a single match with a protein of unassigned function (SWISS-PROT
Q47675
; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in
Saccharomyces cerevisiae
, and in
Caenorhabditis elegans
. The homologous genes (
uppS
) were cloned from
E. coli
,
Haemophilus influenzae
, and
Streptococcus pneumoniae
, expressed in
E. coli
as amino-terminal His-tagged fusion proteins, and purified over a Ni
2+
affinity column. An untagged version of the
E. coli uppS
gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged
E. coli
Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl
2
dependent. With the use of a regulatable gene disruption system, we demonstrated that
uppS
is essential for growth in
S. pneumoniae
R6.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
167 articles.
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