Use of Genomics To Identify Bacterial Undecaprenyl Pyrophosphate Synthetase: Cloning, Expression, and Characterization of the Essential uppS Gene

Abstract

The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC2.5.1.31 ) 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)-[1-3H]-(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. coliproteins 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 inCaenorhabditis 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 Ni2+ 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 MgCl2 dependent. With the use of a regulatable gene disruption system, we demonstrated that uppS is essential for growth in S. pneumoniae R6.

The biosynthesis of peptidoglycan lipid-linked intermediates

“…3). Trace amounts of nona-, deca-, and dodeca-prenyl alcohol derivatives were always detected together with the predominant C 55 -P lipid in membrane extracts, confirming the high but not absolute specificity of the corresponding UppS synthase activities observed during in vitro assays (Thorne & Kodicek, 1966;Higashi et al, 1967Higashi et al, , 1970bScher et al, 1968;Gough et al, 1970;Umbreit et al, 1972;Umbreit & Strominger, 1972b;Apfel et al, 1999).…”

“…The first uppS gene was identified much more recently, in 1998 by Ogura's group, using a genomic DNA library of M. luteus B-P 26 constructed in E. coli and a screening of recombinant clones for overexpression of the synthase activity (Shimizu et al, 1998). Orthologs were subsequently identified in various Gram-positive and Gram-negative bacterial species (Apfel et al, 1999) and this gene was demonstrated to be essential in E. coli (Kato et al, 1999) and Streptococcus pneumoniae (Apfel et al, 1999). Interestingly, these newly identified proteins did not exhibit significant sequence similarity with members of the trans-prenyltransferase family and in particular they did not carry the characteristic aspartate-rich DDXXD motif that is involved in substrate binding via a Mg 21 bridge in the latter enzyme family (Chen et al, 1994).…”

“…Interestingly, these newly identified proteins did not exhibit significant sequence similarity with members of the trans-prenyltransferase family and in particular they did not carry the characteristic aspartate-rich DDXXD motif that is involved in substrate binding via a Mg 21 bridge in the latter enzyme family (Chen et al, 1994). The construction of expression vectors allowed the purification of mg quantities of various UppS in either wild-type or histidine-tagged form (Shimizu et al, 1998;Apfel et al, 1999;Pan et al, 2000). The enzyme activity showed both a detergent (Triton X-100) and MgCl 2 dependency (Apfel et al, 1999), the latter cation being required for the binding and subsequent condensation of isopentenyl pyrophosphate (Chen et al, 2002b).…”

“…The construction of expression vectors allowed the purification of mg quantities of various UppS in either wild-type or histidine-tagged form (Shimizu et al, 1998;Apfel et al, 1999;Pan et al, 2000). The enzyme activity showed both a detergent (Triton X-100) and MgCl 2 dependency (Apfel et al, 1999), the latter cation being required for the binding and subsequent condensation of isopentenyl pyrophosphate (Chen et al, 2002b). UppS has now been characterized in great detail, both biochemically and structurally, by different groups.…”

Manipulation of prenyl chain length determination mechanism of cis‐prenyltransferases

“…showed that not only the primary but also the threedimensional structure of cis-prenyltransferase were totally different from those of trans-prenyltransferases [3][4][5]. Homologous genes for cis-prenyltransferases have been identified in various organisms [6][7][8][9][10][11][12][13], revealing five highly conserved regions in the primary structure of all cis-prenyltransferases [3]. On the other hand, cis-prenyltransferases are classified into three subfamilies with respect to product chain length, i.e.…”