activity of penicillin-binding protein 1a fromThermotoga maritima Julien Offant1,2,3, Mohammed Terrak4, Adeline Derouaux4, Eefjan Breukink5, Martine Nguyen-Diste`che4, Andre´ Zapun1,2,3a
Trang 1activity of penicillin-binding protein 1a from
Thermotoga maritima
Julien Offant1,2,3, Mohammed Terrak4, Adeline Derouaux4, Eefjan Breukink5,
Martine Nguyen-Diste`che4, Andre´ Zapun1,2,3and Thierry Vernet1,2,3
1 CEA, Institut de Biologie Structurale, Grenoble, France
2 CNRS, Institut de Biologie Structurale, Grenoble, France
3 Universite´ Joseph Fourier, Institut de Biologie Structurale, Grenoble, France
4 Centre d’Inge´nierie des Prote´ines, Universite´ de Lie`ge, Institut de Chimie, Sart-Tilman Lie`ge, Belgium
5 Biochemistry of Membranes, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands
Keywords
murein; penicillin-binding protein;
peptidoglycan; screening
Correspondence
T Vernet, Institut de Biologie Structurale, 41
rue Jules Horowitz, 38027 Grenoble, France
Fax: +33 4 38 78 54 94
Tel: + 33 4 38 78 96 81
E-mail: thierry.vernet@ibs.fr
(Received 31 May 2010, revised 22 July
2010, accepted 18 August 2010)
doi:10.1111/j.1742-4658.2010.07817.x
Cell wall biosynthesis is a key target for antibacterial drugs The major constituent of the bacterial wall, peptidoglycan, is a netlike polymer responsible for the size and shape of the cell and for resisting osmotic pres-sure It consists of glycan chains of repeating disaccharide units cross-linked through short peptide chains Peptidoglycan assembly is catalyzed
by the periplasmic domain of bifunctional class A penicillin-binding pro-teins Cross-linking of the peptide chains is catalyzed by their transpepti-dase module, which can be inhibited by the most widely used antibiotics, the b-lactams In contrast, no drug in clinical use inhibits the polymeriza-tion of the glycan chains, catalyzed by their glycosyltransferase module, although it is an obvious target We report here the purification of the ectodomain of the class A penicillin-binding protein 1a from
Thermoto-ga maritima(Tm-1a*), expressed recombinantly in Escherichia coli A deter-gent screen showed that deterdeter-gents with shorter aliphatic chains were better solubilizers Cyclohexyl-hexyl-b-d-maltoside-purified Tm-1a* was found to
be monomeric and to have improved thermal stability A miniaturized, multiwell continuous fluorescence assay of the glycosyltransferase activity was used to screen for optimal reaction conditions Tm-1a* was active as a glycosyltransferase, catalyzing the formation of glycan chains up to 16 disaccharide units long Our results emphasize the importance of the detergent in preparing a stable monomeric ectodomain of a class A pen-icillin-binding protein Our assay could be used to screen collections of compounds for inhibitors of peptidoglycan glycosyltransferases that could serve as the basis for the development of novel antibiotics
Abbreviations
AEC, anion exchange chromatography; C7G, n-heptyl-b- D -glucopyranoside; CMC, critical micellar concentration; CYMAL-4, cyclohexyl-butyl-b- D -maltoside; CYMAL-5, cyclohexyl-pentyl-b- D -maltoside; CYMAL-6, cyclohexyl-hexyl-b- D -maltoside; GTase, glycosyltransferase; IMAC, immobilized metal-ion affinity chromatography; meso-A2pm, meso-diaminopimelic acid; Mtg, membrane-bound monofunctional
glycosyltransferase; PBP, penicillin-binding protein; SEC, size exclusion chromatography; TEV, tobacco etch virus; Tm,melting temperature; Tm-1a* and Tm-GT1a*, ectodomain of Thermotoga maritima penicillin-binding protein 1a and its GTase domain, respectively; TPase, transpeptidase; TSA, thermal shift assay.
Trang 2Peptidoglycan biosynthesis is the major target for
anti-bacterial drugs, such as the most widely used class of
antibiotics, the b-lactams, or the last-resort
glycopep-tides (vancomycin) Peptidoglycan is a strong, netlike
polymer responsible for maintaining the shape of the
bacterial cell and resisting intracellular osmotic
pres-sure It consists of glycan chains of repeating
disaccha-ride units cross-linked through short peptide chains
[1,2] Peptidoglycan biosynthesis remains a major
tar-get for new antibiotics, as it is both unique and
essen-tial to bacteria
The final steps of peptidoglycan assembly are
cata-lyzed by penicillin-binding proteins (PBPs), which are
membrane-bound enzymes exposed to the
extracyto-plasmic medium For this reason, PBPs are the most
interesting targets among the enzymes involved in the
biosynthesis of the peptidoglycan, as they are easily
accessible to drugs
In addition to a short N-terminal cytoplasmic region
and a transmembrane segment, high molecular mass
class A PBPs possess two enzymatic modules, a
glyco-syltransferase (GTase) and a transpeptidase (TPase)
[3] The former is responsible for the elongation of the
glycan strands from the precursor lipid II, a
disaccha-ride (MurNAc-b-1,4-GlcNAc) pentapeptide anchored
to the membrane by a C55 undecaprenyl chain via a
pyrophosphate The latter module cross-links the
pep-tide chains by a transpeptidation reaction [1,3,4] The
extracytoplasmic region of high molecular mass class B
PBPs harbors an N-terminal module of unknown
func-tion and a TPase module, but lacks the GTase module
Low molecular mass PBPs contain only a single TPase
domain, but with endopeptidase or carboxypeptidase
activity Finally, membrane-bound monofunctional
GTases (Mtgs) have also been identified in a few
bac-teria [5–8] Mtgs and GTase modules of class A PBPs
belong to family 51 of glycosyltransferases from the
CAZy database [9,10]
Penicillin and other b-lactam antibiotics are specific
inhibitors of TPase activity After six decades of
b-lac-tam use, bacterial pathogens are now widely resistant
to this most broadly used class of drug [11]
GTase activity is an obvious promising target for
the development of new antibiotics, but despite many
years of effort, no drug candidate is currently available
for clinical use Moenomycin was isolated from
Strep-tomyces ghanaensisin 1968 [12], and is the
best-charac-terized natural antibiotic that directly inhibits GTase
activity This phosphoglycolipid inhibits GTases at
nanomolar concentrations, and has strong antibiotic
activity in vitro It is not used in human medicine,
because of poor pharmacokinetics and oral bioavail-ability [13] In addition, Gram-negative bacteria are not susceptible to moenomycin, as it cannot penetrate the outer membrane of these organisms [14]
Several crystal structures of GTase are now avail-able, with and without bound moenomycin or deriva-tives: the extracellular domain of PBP2 from Staphylococcus aureus [15,16] and of the Mtg from the same organism [17], the GTase module of PBP1a from Aquifex aeolicus [18,19], and the full-length PBP1b from Escherichia coli [20]
For a long time, the functional characterization of the GTase activity of class A PBPs or Mtgs has been limited to basic studies by the lack of availability of the lipid II substrate and analogs [21] The development of chemically synthesized [22,23] and enzymatically syn-thesized [24,25] lipid II has allowed the development of various in vitro GTase activity assays Significant mech-anistic insights have been obtained [8,26–33] In partic-ular, the direction of the glycan chain elongation has been established, where a disaccharide unit is added to the reducing end of the growing chain, using appropri-ately blocked substrate analogs [31,34] Also, different GTases produce chains with different length distribu-tions [8] Screening methods for the identification of GTase inhibitors have been proposed [35–37]
As targets of potential new antibacterial drugs, it is desirable to study a greater variety of peptidoglycan GTases from various organisms We report here the development of a multiwell assay, based on the method
of Schwartz et al [26], to screen for optimal reaction conditions that may differ from enzyme to enzyme The method is exemplified by a study of the full ectodomain of PBP1a from Thermotoga maritima (Tm-1a*), extending our previous work on the GTase domain from this protein (Tm-GT1a*) [38] T maritima
is a Gram-negative hyperthermophilic bacterium isolated from hot sea floors [39] The fact that proteins from hyperthermophilic organisms are often good candidates for crystallographic structural studies [40] prompted us
to purify Tm-1a* in an active form The properties of the purified enzyme have been determined and com-pared with those of other GTases
Results and Discussion
The solubilization efficiency of Tm-1a* is related
to the detergent alkyl chain length Tm-1a* has been defined on the basis of sequence alignments, and spans residues Glu34–Gly643 The
Trang 3N-terminal region thus defined is likely to include two
short b-strands (b1 and b2) that are now known to be
part of a five-stranded b-sheet, with other strands
being contributed by the TPase domain at the
interdo-main junction [15,41] An alignment with sequences of
known crystal structure (S aureus PBP2 and A
aeoli-cus PBP1a) is shown in Fig S1 The sequence of
T maritima PBP1a with the predicted domains is
shown in Fig S2 The sequence identities of T
mariti-maPBP1a with S aureus PBP2 and A aeolicus PBP1a
are 21% and 25% respectively; for the GTase domain,
the identities are 32% and 35%, respectively
Protein expression was performed in E coli
BL21(DE3) CodonPlus-RIL to favor translation of the
numerous rare codons (15%) of the Tm-1a* sequence
Initial purification attempts were performed in the
presence of 1% of the zwitterionic Chaps detergent, as
performed previously for the isolated GTase domain
[38] This allowed solubilization of about 80% of the
expressed Tm-1a* However, immobilized metal-ion
affinity chromatography (IMAC)-purified Tm-1a* in
Chaps was partially aggregated, as shown by size
exclusion chromatography (SEC) analysis (data not
shown), as was observed with the isolated GTase
domain [38]
To solve this problem, we compared the
solubiliza-tion efficiency of 11 detergents Tm-1a* was solubilized
from intact bacteria by sonication in the various
deter-gent solutions prior to purification with an Ni2+
–nitrilo-triacetic acid Superflow column and SDS⁄ PAGE
analysis (Fig 1) The maximal yield of purification was
obtained with Chaps followed by
n-heptyl-b-d-gluco-pyranoside (C7G) and cyclohexyl-hexyl-b-d-maltoside
(CYMAL-4) Interestingly, the solubilization efficiency
was related to the length of the carbon chain: the shorter
the chain, the better the solubilization For instance, the
solubilization efficiency of Tm-1a* with CYMAL-4
was 40%, which was 70% higher than with
cyclohexyl-pentyl-b-d-maltoside (CYMAL-5) and
cyclohexyl-hexyl-b-d-maltoside (CYMAL-6), respectively This pattern
was also found for the three alkyl-glucoside and the two
alkyl-maltoside detergents CYMAL-4 was chosen for
further comparison with Chaps
Purification of monomeric CYMAL-4-solubilized
Tm-1a*
Bacteria expressing Tm-1a* were lysed by sonication
in the presence of 0.74% CYMAL-4 The first IMAC
procedure delivered a protein over 80% pure
(Fig 2A) Most contaminants were eliminated by
pre-parative SEC The N-terminal His6-tag was cleaved by
the His6-tagged tobacco etch virus (TEV) protease,
and uncleaved Tm-1a*, the free tag and the protease were retained on the Ni2+–nitrilotriacetic acid column
A final anion exchange chromatography (AEC) step removed trace contaminants CYMAL-4-purified Tm-1a* was eluted as a single symmetrical peak on an analytical SEC column (Fig 2B), with an apparent molecular mass of 72 kDa (theoretical molecular mass
of 69 700 Da) The higher apparent molecular mass
is probably attributable to the presence of bound CYMAL-4 molecules SDS⁄ PAGE of the SEC peak showed a highly homogeneous protein (Fig 2B, insert) From 1 L of E coli culture, 0.5 mg of pure and homogeneous Tm-1a* monomer was obtained The protein concentrated to 4.4 mgÆmL)1 (60 lm) remained monomeric over time at 4C and )80 C The TPase domain of Tm-1a* was functional for reac-tion with b-lactams, as its transpeptidase site could be labeled with fluorescent ampicillin (Fig 2C)
CYMAL-4-solubilized Tm-1a* displays elevated thermal stability
The thermal shift assay (TSA) is an efficient and easy way to measure the thermal stability of proteins as compared with other biophysical methods, such as CD and microcalorimetry The method is also amenable to
Fig 1 Detergent screening for the recovery of Tm-1a* Lysates of
E coli cells overexpressing Tm-1a* were prepared in the presence
of various detergents at concentrations twice their CMCs and loaded onto an Ni 2+ IMAC column (A) SDS ⁄ PAGE of IMAC-eluted fractions, Coomassie-stained 72 kDa Tm-1a* band (B) Histogram
of the intensity of the Tm-1a* bands relative to that obtained with Chaps Black or shades of gray denote detergents of the same chemical family C8G, n-octyl-b- D -glucopyranoside; C9G, n-nonyl-b- D -glucopyranoside; C 10 M, n-decyl-b- D -maltopyranoside; C 12 M, n-dode-cyl-b- D -maltopyranoside; LDAO, lauryldimethylamine-oxide.
Trang 4high throughput, and this allows the screening of
com-pounds or conditions (pH, ionic strength, etc.) [42,43]
that influence protein stability The TSA is more
diffi-cult to implement on detergent-solubilized proteins
than without detergent The fluorescence of the
Sypro-Orange probe increases when the molecule is in contact
with a nonpolar environment such as exposed hydro-phobic residues The detection of a fluorescence increase upon denaturation of a protein will depend on the number and hydrophobicity index of exposed resi-dues during unfolding The micellar phase of a deter-gent can have a similar effect, complicating the TSA results
With Chaps-purified Tm-1a*, two denaturation tran-sitions (Tm) were measured at 61 ± 1 and 81 ± 1C These two transitions might correspond to independent unfolding of the GTase and TPase domains The Tm
of Chaps-purified Tm-GT1a* was determined to be
62 ± 1C It is therefore tempting to attribute the lower and higher Tm values to the unfolding of the GTase and TPase domains, respectively If this inter-pretation is correct, the TPase domain is more stable than the GTase domain by about 20C, and the TPase domain within Tm-1a* does not influence the stability of the GTase domain The crystal structure of PBP2 from S aureus [15] shows a narrow neck con-necting, with some flexibility, the GTase and TPase domains, with little contact between the domains This structure is consistent with the absence of mutual sta-bilizing effect of the domains
The thermal stability of CYMAL-4-purified Tm-1a* also displays two thermal transitions at the higher values of 79 ± 1 and 89 ± 1C These values are compatible with a thermophilic origin of the protein CYMAL-4-purified Tm-1a* is more stable than Chaps-purified Tm-1a* This observation, together with the fact that CYMAL-4-purified Tm-1a* does not aggregate, led us to select CYMAL-4 for purification and storage of Tm-1a*
Screening of reaction conditions of Tm-1a* in a multiwell plate format
In vitro GTase activity is strongly influenced by the nature and concentration of additives, including deter-gent, dimethylsulfoxide, or metal [26] We have minia-turized in a multiwell format the continuous fluorescence assay described by Schwartz (2002) This allows the parallel screening of numerous conditions while reducing the use of the limiting reagent lipid II The assay takes advantage of the higher fluorescence
of dansylated lipid II solubilized by detergent micelles than of the free dansylated pentapeptide disaccharide GTases polymerize glycan chains by transferring the growing chain from its undecaprenyl pyrophosphate anchor onto an additional lipid II unit [31] Following the hydrolytic action of the muramidase included in the reaction mix, assembled glycan chains are degraded into pentapeptide disaccharide units As the fluorescence
Fig 2 Purification of CYMAL-4-solubilized Tm-1a* (A)
Coomassie-stained SDS ⁄ PAGE of the initial Ni 2+ IMAC purification: m, molecular
mass markers; L, clarified lysate loaded; FT, flowthrough; 1–4,
elution fraction with 250 m M imidazole *The 72 kDa Tm-1a* band.
(B) Analytical SEC (Superdex-200 HR 16⁄ 30; GE Healthcare) of
Tm-1a* purified on Ni2+IMAC followed by SEC and AEC (C) SDS ⁄
PAGE of Tm-1a* labeled with fluorescein–ampicillin (25 l M for
10 min at 37 C): 1, after Coomassie blue staining; 2, fluorescence
image taken with MOLECULAR IMAGER (Bio-Rad).
Trang 5of the soluble dansylated pentapeptide disaccharide is
lower than that of the dansylated lipid II, the GTase
activity can be followed as a decrease of dansyl
fluo-rescence
Our multiwell version of the assay is initiated by the
addition of the GTase prior to measurement of the
flu-orescence over the time course, and allows the parallel
monitoring of up to 96 reactions Visualization of the
time courses allows easy comparison of the various
reactions (Fig 3) and an initial selection of the
opti-mal conditions GTase activities were compared by
measuring the initial rate of fluorescence decrease
(Fig 3)
The optimal conditions that produced the highest
initial slope representing the fastest incorporation of
lipid II into glycan chains contained 20%
dimethylsulf-oxide and decyl-poly(ethylene glycol) at 2.5-fold or
5-fold the critical micellar concentration (CMC)
Almost equally good were conditions with 5–10%
dimethylsulfoxide and decyl-poly(ethylene glycol) at its
CMC The reaction was completely inhibited in the
presence of 10 lm moenomycin (Fig 3)
The specific activity of Tm-1a* was determined in
the presence of 2 lm [14C]lipid II to be 2.9 ± 0.5 nmol
of lipid II used min)1ÆmgÆenzyme)1, which is about
10-fold less than that of E coli PBP1b (25 ± 5 nmol of
lipid II used min)1ÆmgÆenzyme)1) However, this might
reflect the fact that the optimal temperature is
proba-bly higher for a Tm-1a* that originates from a
thermo-philic organism
Determination of the glycan chain size distribution of Tm-1a* products The SDS⁄ PAGE assay developed by Barrett et al [30] allows visualization of the length of the synthesized glycan chains, using radiolabeled lipid II as precursor Tm-1a* was found, in our experimental conditions, to produce rather short chains, with the main product being about 10 disaccharide units long, without detect-able chains longer than 16 units (Fig 4) PBP1b from
E coli was shown to produce longer chains under the same conditions (about 30 units long), indicating that Tm-1a* is less processive Of the GTases tested so far, Tm-1a* appears to be the one that synthesizes the shortest chains [8] Therefore, this enzyme could be useful for applications such as generation of glycan chains with defined size and composition that can be used as substrates for other enzymes (peptidoglycan hydrolases, sortases, or modifying enzymes, such as amidases and muramidases) or as a molecular stan-dards in SDS⁄ PAGE analysis
It must be noted that the substrates used in this study were either l-lysine-containing lipid II,
dansylat-ed on the lysine, or radiolabeldansylat-ed meso-diaminopimelic acid (meso-A2pm)–lipid II It has been found recently that the T maritima peptidoglycan contains unusual stem peptides, which include d-lysine in nonconven-tional arrangements, as well as unusual cross-links [44] Future studies will investigate the substrate speci-ficity of the TPase reaction of Tm-1a*
Fig 3 Screening of conditions for GTase activity of CYMAL-4-purified Tm-1a* Tm-1a* (250 n M ) was incubated in the presence of dansylated lipid II (10 l M ) in
50 m M Hepes (pH 7.5), 200 m M NaCl,
10 m M CaCl2and muramidase (1 unit) with various combinations of decyl-poly(ethylene glycol) and dimethylsulfoxide
concentrations Fluorescence (excitation at
340 nm and emission at 520 nm) was monitored for 80 min The best reaction conditions, highlighted in gray, show the greatest initial slopes Complete inhibition with 10 l M moenomycin is presented below
as a negative control.
Trang 6GTases that participate in peptidoglycan assembly
are obvious targets for the development of novel
anti-biotics To this end, it will be necessary to study
multi-ple GTases from diverse microorganisms We have
reported here a preliminary characterization of
Tm-1a* It will be of interest to similarly prepare and
characterize the full-length protein, to probe the
influ-ence of the transmembrane segment Also, T maritima
is hyperthermophilic, and future studies of T maritima
PBP1a should investigate how the kinetics and
proces-sivity vary with temperature Our results emphasize the
importance of the detergent in preparing a stable
monomeric ectodomain of a class A PBP Optimal
reaction conditions for the polymerization of glycan
chains by Tm-1a* were found with the use of a
multi-well assay This assay could be used to screen
collec-tions of compounds for inhibitors of peptidoglycan
glycosyltransferases that could serve as a basis for the
development of novel antibiotics
Experimental procedures
Gene cloning and protein expression
The fragment encoding the extracellular region of PBP1a
(accession number AAD35967.1) was PCR-amplified from
T maritimaMSB8 genomic DNA with the forward primer 5¢-GAAAATCTGTATTTTCAGGGCGAGGAGAAACT TGTGCCGACC-3¢ and the reverse primer 5¢-TCACCAT CCAATTGATTAACCTCCTTCCATCAAAAACTTTTT CCAGATTTC-3¢ The fragment coding for the isolated GTase was PCR-amplified with the same forward primer and the reverse primer 5¢-TCACCATCCAATTGATTA TTCCGCAGAGTAATTCTCGTATTCCTG-3¢ (sequences required for ligation-independent cloning are underlined) Purified PCR products were introduced into pLIM01 [45]
by the ligation-independent cloning method [46], to produce the pTm-1a* and pTm-GT1a* expression vectors encoding Tm-1a* (Glu34–Gly643) and the corresponding GTase domain (Glu34–Thr244) with an N-terminal His6-tag fol-lowed by the TEV protease cleavage site
E coli BL21-CodonPlus(DE3)-RIL cells (Stratagene, Cedar Creek, TX, USA) were transformed with pTm-1a* and pTm-GT1a*, and overnight precultures were diluted 50-fold into fresh LB medium supplemented with antibiot-ics After growth at 37C to an attenuance of 0.8 at
600 nm, protein expression was induced with 0.5 mm iso-propyl-thio-b-d-galactoside, and incubation was continued overnight at 15C
Detergent screening for Tm-1a* solubility and purification by Ni2+IMAC
Detergent screening was performed from 10 mL aliquots of induced bacterial cultures at an attenuance of 4 (600 nm), spun, and resuspended in 1.2 mL of lysis buffer (25 mm Hepes, pH 7.5, 500 mm NaCl) containing the appropriate detergents at a concentration twice their CMC The 11 fol-lowing detergents were used: Chaps, 1%; lauryldimethyl-amine-oxide, 0.0064%; Triton X-100, 0.03%; C7G, 3.8%; n-octyl-b-d-glucopyranoside, 1.37%; nonyl-b-d-glucopyr-anoside, 0.4%; decyl-b-d-maltopyrnonyl-b-d-glucopyr-anoside, 0.174%; n-dodecyl-b-d-maltopyranoside, 0.0174%; CYMAL-4, 0.74%; CYMAL-5, 0.24%; and CYMAL-6, 0.056% (all purchased from Anatrace, Maumee, OH, USA) Samples of 400 lL of the resuspension were placed in 1.1 mL MicroTubes (National Scientific Supply, San Rafael, CA, USA) and simultaneously lysed by sonication, using a microplate horn coupled with an S3000 generator (Misonix, Newtown, CT, USA) The horn was filled with ice-cold water, and cells were lysed by six pulses of 1 min with 2 min intervals (power level set to 7)
Lysis supernatants (20 000 g, 30 min, 4C) were loaded onto 50 lL of Ni Sepharose High Performance resin pre-packed in a 96-well format His MultiTrap HP (GE Health-care, Little Chalfont, UK) After a wash with 1 mL of
60 mm imidazole in the same buffers, Tm-1a* was eluted with 100 lL of 250 mm, imidazole and eluates were analyzed
by SDS⁄ PAGE Detergents were evaluated by quantifying the Coomassie-stained bands corresponding to Tm-1a* with
a BioRad Geldoc 2000 (BioRad, Hercules, CA, USA)
Fig 4 Glycan chain size distribution of Tm-1a* products An
auto-radiogram of SDS⁄ PAGE-separated glycan chains is shown GTase
reaction products show that the maximal length of the glycan
chains made by Tm-1a* is about 16 disaccharide units (n), with the
main product being 10 units long As a control, glycan chains
poly-merized in similar conditions by E coli PBP1b are shown.
Trang 7Purification of Tm-GT1a* and Tm-1a*
For the purification of CYMAL-4-solubilized Tm-1a*, a
1 L culture at a final attenuance at 600 nm of 4 was spun
and resuspended in 90 mL of 25 mm Hepes (pH 7.5),
500 mm NaCl, 0.74% CYMAL-4 and a pill of Complete
EDTA-free protease inhibitors (Roche, Basel, Switzerland)
Cells were lysed using a 1⁄ 2 inch horn coupled with an
S3000 generator (Misonix), with a total sonication time of
4 min (2 s pulses at 10 s intervals) and a power level of 5
After centrifugation (20 000 g, 30 min, 4C), the
superna-tant was loaded onto 5 mL of Ni2+–nitrilotriacetic acid
Su-perflow (Qiagen) pre-equilibrated with buffer A (25 mm
Hepes, pH 7.5, 500 mm NaCl, 0.37% CYMAL-4) Tm-1a*
was eluted with two steps of 60 mm and 250 mm imidazole
in buffer A Further purification steps consisted of SEC on
a Superdex-200 preparation grade column (GE Healthcare)
equilibrated with buffer A, IMAC following cleavage of
the His6-tag by the TEV protease (to remove noncleaved
Tm-1a* and the protease), and AEC on a 1 mL
Resour-ce Q column (GE Healthcare) eluted with a 0–1 m
linear gradient in buffer A to remove trace contaminants
CYMAL-4-purified Tm-1a* (0.2 mgÆmL)1) was frozen in
liquid nitrogen, and stored at)80 C
A similar protocol was adopted for the purification of
Chaps-solubilized Tm-GT1a* and Tm-1a*, but omitting the
AEC Cell lysis was performed with 1% Chaps, and all
sub-sequent buffers contained 0.7% Chaps Final
concentra-tions were 0.5 mgÆmL)1 for Tm-GT1a* and 0.4 mgÆmL)1
for Tm-1a* Purified enzymes were stored at)80 C
TSA
Experiments were carried out using the IQ5 96-well format
real-time PCR instrument (BioRad) Briefly,
CYMAL-4-purified Tm-1a* (60 lm) and Chaps-CYMAL-4-purified Tm-GT1a* or
Tm-1a* at concentrations ranging from 15 to 40 lm were
mixed with 2 lL of 100-fold water-diluted 5000X SYPRO
Orange (Molecular Probes, Eugene, Oregon, USA)
Sam-ples were heat-denatured from 20 to 100C at a rate of
1CÆmin)1, and unfolding was monitored by measuring
changes in the fluorescence of SYPRO Orange The Tm
val-ues were identified as the maxima of the first derivatives of
the fluorescence versus temperature curves
Detergent-con-taining buffers were used as blanks, and their SYPRO
Orange fluorescence curves were subtracted from the
sam-ple curves
Screening of conditions for the GTase activity
The assay developed by Schwartz [26] was adapted to a
96-well format with a medium binding black 96-well
microplate (Greiner Bio One, ref 655076; Frieckenhausen,
Germany) in a FLUOstar OPTIMA Microplate reader
(BMG Labtech, Offenburg, Germany) The reaction mix
(50 lL) included lysine-dansylated lipid II (10 lm) [24] in
50 mm Hepes (pH 7.5), 200 mm NaCl, 10 mm CaCl2, 1 unit
of N-acetylmuramidase from Streptococcus globisporus (Cal-biochem, Darmstadt, Germany), and various combinations
of decyl-poly(ethylene glycol) (0, 1, 2.5, 5, 10, 20 and
40· CMC) and dimethylsulfoxide (0%, 5%, 10% and 20%) concentrations Time courses at 30C were initiated with the addition of Tm-1a* (250 nm) and followed for 80 min with excitation at 340 nm and emission recorded at 520 nm
Determination of specific GTase activity of Tm-1a*
The GTase assay was carried out in triplicate, using the [14C]meso-A2pm–lipid II (2 lm; 0.126 lCiÆnmol)1) as sub-strate in the following reaction mix: 50 mm Hepes (pH 7.5),
10 mm CaCl2, 200 mm NaCl, 0.2% decyl-poly(ethylene gly-col) and 20% dimethylsulfoxide The reaction was started with addition of 150 nm Tm-1a* and stopped with 12.5 lm moenomycin (Flavomycin; Hoechst, Frankfurt, Germany) The reaction products were separated by TLC in
propanol-2⁄ ammonia ⁄ water (6 : 3 : 1), and analyzed with a Molecu-lar Imager (BioRad)
Determination of the glycan chain size distribution of Tm-1a* products
The reaction conditions were: 50 mm Hepes (pH 7.5),
10 mm CaCl2, 200 mm NaCl, penicillin G 1000 UÆmL)1 (500 lgÆmL)1), 0.2% decyl-poly(ethylene glycol), 20% dimethylsulfoxide, 4 lm [14C]meso-A2pm-lipid II (0.126 lCiÆnmol)1), and 2 lm Tm-1a* or 0.5 lm E coli PBP1b Samples were collected at various times and analyzed by SDS⁄ PAGE 9%T 2.6%C (size 20 cm · 20 cm · 1 mm), with an anode buffer of 0.1 m Tris (pH 8.3) and a cathode buffer of 0.1 m Tricine (pH 8.45) and 0.1% SDS [30]
Acknowledgements
This work was partly funded by the FP6 EUR-INTA-FAR LSHM-CT-2004-512138 project and the ANR grant PneumoPG ANR-08-BLAN-0201 We thank B Gallet and M Noirclerc-Savoye, from the IBS plat-form of the Partnership for Structural Biology and the Institut de Biologie Structurale in Grenoble (PSB⁄ IBS), for their expertise with the fluorimeter
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Supporting information
The following supplementary material is available: Fig S1 Sequence alignment of class A PBPs
Fig S2 Sequence of T maritima PBP1a with pre-dicted domains
This supplementary material can be found in the online version of this article
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