Chemistry & Biology
CDP-ME Kinase from Bacterial Pathogens
EXPERIMENTAL PROCEDURES
log (MSMEG_5436) replacement plasmid (pPR27::Sm ispE::KanR) and
a rescue plasmid (pCG76-Sm ispE) is illustrated in Figure 3A. Briefly, a 2849
bp DNA fragment containing M. smegmatis ispE ortholog (MSMEG_5436),
940 bp upstream, and 940 bp downstream of the gene was amplified by
PCR using the MutSmispE-F and R (Table S3). The DNA fragment was
cloned to pGEM T plasmid and a 1250 bp Kan resistance cassette (KanR)
was cut out with HincII from plasmid pUC4K, and inserted into the Klenow
fragment (Invitrogen)-filled RsrII site of the MSMEG_5436 gene. A 2849 bp
SM ispE::KanR fragment was cut by SpeI and XbaI and moved to the same
enzyme site of pPR27 (temperature-sensitive mycobacterial origin of replica-
tion; sacB, xylE, gentamicin resistant) (Pelicic et al., 1997), yielding pPR27::SM
ispE::KanR, the vector used to achieve homologous recombination event
at the SMEG_5436 locus of M. smegmatis. The pPR27 used in this study
has the mycobacterial temperature-sensitive origin of replication. Thus,
pPR27::SM ispE::KanR can replicate at 32ꢀC but is efficiently lost at 42ꢀC
and above (Pelicic et al., 1997; Pan et al., 2001). In addition, the plasmid
also contains the counterselectable marker sacB from Bacillus subtilis (Pelicic
et al., 1997) for use in selection of the double-crossover event in the presence
of sucrose.
Materials
Genomic DNA and a cytosolic fraction isolated from wild-type M. tuberculosis
H37Rv were provided by TB Vaccine Testing and Research Materials Contract,
Colorado State University (NIH NIAID N01-AI-40091). S. Typhi (700931D) and
V. cholerae (39315D) genomic DNA were purchased from the American Type
Culture Collection (ATCC, Manassas, VA). B. mallei genomic DNA was kindly
provided by Dr. Herbert Schweitzer (Colorado State University). All PCR
reagents and cloning materials were purchased from QIAGEN (Valencia,
CA). His-select nickel affinity resin, ATP, and reagents were obtained from
Sigma-Aldrich (St. Louis, MO). [g-32P]ATP (6000 Ci/mmol) and PD-10 columns
were purchased from Amersham Biosciences (Pittsburgh, PA). The ADP Quest
HS Kinase Assay Kit was purchased from GE Healthcare Bio-Sciences Corp.
(Piscataway, NJ). All other reagents and solvents were of at least analytical
grade.
Synthesis of Enantiopure CDP-ME
Approximately 0.54 mmol tertiary alcohol 3 was dissolved in 10 ml freshly
distilled dry tetrahydrofuran (THF) and cooled to 0ꢀC. To the cooled solution,
1.08 mmol potassium hydride was added and stirred for 30 min at room
temperature. The reaction mixture was cooled to 0ꢀC again and 1.35 mmol
benzyl bromide was added slowly, warmed to room temperature, and stirred
for an additional 2 hr. The reaction was quenched by addition of saturated
ammonium chloride solution and extracted with ethyl acetate. The crude
mixture was purified by silica gel column chromatography using ethyl acetate:
hexane (7:3, v/v) as the solvent, yielding 0.486 mmol benzylated product (90%
yield) 4.
The entire M. smegmatis MESG_5436 gene was amplified by PCR with using
primer set (SmispE-F and R) (Table S3) and cut with NdeI and BamHI and
ligated into the same enzyme sites downstream of the heat shock promoter
Phsp60 in plasmid pCG76 (E. coli/Mycobacterium shuttle vector carrying
a temperature sensitive origin of replication, and streptomycin resistance),
yielding pCG76::SM ispE (Figure 3A). Plasmid pCG76 carries the same
temperature-sensitive mycobacterial replication origin as pPR27, and thus
can replicate at the permissive temperature of 32ꢀC but is abrogated at
42ꢀC and above (Jackson et al., 2000).
Data for 3: 1H-NMR (CDCl3, 300 MHz): d 7.34 (m, 10H), 5.78 (d, 1H, J =
3.6 Hz), 5.02-5.09 (m, 4H), 4.03-4.26 (m, 3H), 3.96 (dd, 1H, J = 2.7 & 7.8 Hz),
1.56 (s, 3H), 1.36 (s, 3H), 1.12 (s, 3H). 13C-NMR (CDCl3, 75 MHz):135.7,
135.6, 128.6, 128.5, 128.4, 128.0, 127.9, 112.7, 103.4, 83.9, 80.2, 76.1, 69.4,
69.3, 65.9, 26.5, 18.1.; IR (neat, cm-1): 3588, 2966, 2362, 2336, 1652, 1614.
HRMS (ESI) C23H30O8P (M+H+): calculated 465.1673 and found 465.1680;
[a]D 33.0 (c 0.5, CHCl3).
Data for 4: 1H-NMR (CDCl3, 300 MHz): d 7.34 (m, 15H), 5.80 (d, 1H, J =
3.6 Hz), 5.02-5.07 (m, 4H), 4.54-4.67 (m, 2H), 4.08-4.35 (m, 4H), 1.60 (s, 3H),
1.37 (s, 3H), 1.20 (s, 3H). HRMS (ESI) C30H36O8P (M+H+): calculated
555.5758 and found 555.5762 ; [a]D 13.0 (c 0.3, CHCl3).
PCR Amplification of Putative ispE Orthologs of M. tuberculosis,
B. mallei, S. Typhi, and V. cholerae
Homologs of E. coli IspE from the genomes of bacterial pathogens were iden-
tified by sequence alignment. The M. tuberculosis H37Rv genome is available
three bacterial genome sequences are available on the GenBank of National
for amplification of putative ispE open reading frames. The PCR primers
(Table S3) were designed to contain NdeI or HindIII restriction enzyme sites
in the forward and reverse primers, respectively.
For MEP and CMP coupling, 0.15 mmol CMP was dissolved in 1 ml aceto-
nitrile followed by addition of 0.6 mmol N,N, dimethyl aniline and 0.15 mmol
triethylamine at 0ꢀC. Then, 0.76 mmol trifluoroacetic anhydride in acetonitrile
was slowly added to the mixture and stirred for 15 min. Excess TFA and anhy-
dride was removed under reduced pressure. To the above reaction mixture,
a combination of 0.45 mmol 1-methyl imidazole and 0.76 mmol triethylamine
in acetonitrile was added slowly and stirred for 30 more min. The activated
CMP was added to the mixture of 0.125 mmol MEP and activated 4 Aꢀ molec-
ular sieves in acetonitrile at 0ꢀC and stirred for 2 hr. After the reaction was
completed, the mixture was extracted with chloroform and the aqueous
layer was lyophilized. CDP-ME was purified by sequential chromatography
on a Bio-Gelꢀ P-2 gel fine column using 100 mM aq. ammonium bicarbonate
followed by further purification on a benzyl DEAE cellulose anion exchange
column, using a gradient of 0 to 0.5 M aq. ammonium bicarbonate solution.
This resulted in a 45% yield of CDP-ME.
Cloning of Eight M. tuberculosis ispE Fragments
Purification of the native form of the putative M. tuberculosis IspE (Rv1011)
proved difficult as reported previously (Sgraja et al., 2008); however, soluble
M. tuberculosis IspE was obtained by removal of amino acid residues at either
the N terminus, C terminus, or both termini. Genetic modifications were
designed using conserved domain searches for M. tuberculosis IspE as a query
performed by tools available in the NCBI (specialized BLAST) (Figure 4B). The
PCR products of eight Rv1011 fragments were amplified using the primer sets
shown in Table S3. For amplification of Rv1011 fragments I through VIII
(Figure 4B), primers were designed based on the sequences available in
TubercuList (Table S3) and were synthesized by Macromolecular Resources
(Colorado State University). The eight amplified PCR products were digested
with NdeI and HindIII, and ligated into the pET28a(+) vector (EMD Biosciences,
Inc., San Diego, CA). The ligation mixtures were used to transform the E. coli
DH5a cloning host (Life Technologies, Rockville, MD), generating DH5a
[pET28a(+):: Rv1011 I] through DH5a[pET28a(+):: Rv1011 VIII]), which were
subsequently purified using Gene Extraction Kits (QIAGEN, Valencia, CA)
and sequenced by Macromolecular Resources.
Data for CDP-ME: 1H-NMR (D2O, 300 MHz): d 7.94 (d, 1H, J = 7.5 Hz), 6.09
(d, 1H, J = 7.5 Hz), 5.96 (d, 1H, J = 4.2 Hz), 4.27 (m, 6H), 3.95 (m, 1H), 3.80
(d, 1H, J = 10.5 Hz), 3.57 (d, 1H, J = 11.7 Hz), 3.44 (d, 1H, J = 11.7 Hz), 1.10
(s, 3H). HRMS (ESI) C14H25O14N3P2 (M-H+) calculated 520.0728 and found
520.0724; [a]D 13.7 (c 0.2, H2O).
Construction of Vectors for the Determination of Mycobacterial
IspE Essentiality
Cloning of Truncated ispE Orthologs of B. mallei, S. Typhi,
and V. cholerae
Initially, attempts were made to demonstrate ispE essentiality in M. tubercu-
losis using previously reported methods (Eoh et al., 2007; Parish and Stoker,
2000); however, attempts to obtain a chromosomal deletion in the merodiploid
background (complemented) were unsuccessful, due to unidentified technical
difficulties. Thus, we utilized allelic disruption to demonstrate essentiality in the
related species, M. smegmatis. The construction of M. smegmatis ispE ortho-
Forward and reverse primers for amplifying ispE of B. mallei, S. Typhi, and
V. cholerae were designed to remove the five C-terminal amino acids
(Table S3), resulting in generation of three truncated versions of bacterial
IspE enzymes. Expression constructs, pET28a(+)::mBME, pET28a(+)::mSTE,
and pET28a(+)::mVCE were generated as described above.
Chemistry & Biology 16, 1230–1239, December 24, 2009 ª2009 Elsevier Ltd All rights reserved 1237