1. INTRODUCTION
acctyltransfcraxe (CAT; EC Chloramphcnicol 2.3.1.28) is a homotrimer which catalyses the acccylCo&dependent O-acetylation and inactivation of chloramphcnicol, a modification which confers resistance to the antibiotic on host cells [l]. From the three-dimensional X-ray crystal structure of the type III variant of CAT (CATIII), it is known that N3 of the imidazolc ring of H 195 is suitably placed to act as the proposed base catalyst in the reaction [2,3]. Study of the histidinc C2-‘H rcsonanccs in CAT is complicated by its overall molecular mass incorporation in combination with single and multiple quantum coherence experiments has been particularly valuable in the simplification of ‘H spectra of smaller proteins by [4-T]. We sow here that these methods permit detection of histidine C2-‘H resonances even in proteins as large as CAT. 2. EXPERIMENTAL [ri,fg2-“C’JL-Mistidine at 92% “C was obtained from Fluorochem (Old Glossop, Derbyshire, UK) and a 62% “C from Drs R. Gigg and J. Feeney (NIMR, Mill Hill, London). An E, coli histidine auxotroph (strainJMl100 from Dr R.A. Cooper [S]) was used to selectively incorporate labclled histidine into CAT. Transformation with the
Correspondence address: J.P. Derrick, Department t.hiVerSi~y
Gf
Abbrevlalions: heteronuclear
Published
Lcicestcr, CAT, multiple
of Biochemistry,
Leicester LE? 9!?Hi 1JK chloramphenicol acetyltransferase; quantum coherence
by Elsevier Science Publishers
B. V.
HMQC,
in 6. coii 191, wzu eonduclcd us/& i\ CpCl~ ~ro&iure [LO). Optimai growth conditions for production of CATIII were round to be in n medium containing (in g/l), Nn#PQ.t 6.8, KHzP& 3.0, NaCI 0.5, NH& 1.0, MyS& lI.49, thymine 0. I, glycerol 4.0, I.-arpininc, 0,28, I--uspnraric acid 0.74, L-cystcineO.20, L.glycirrr 0.20, L-glurnmicacid 2.8, L-isolcucinc 0S54, L-l~ucine 0.70, L-lyxine 0.74, L-mcthioninc 0.20, L.~p~ieriylnlatii~ic O.LJ, L.nrolinc 0.80. L-rhrconinc 0.60. Ltryptophjn 0[26, L-ryrosine’0.62, L-valinc d-82, L-&nine 0.3j, LJerinc 0.20, I,-asparnginc 0,23, L&utaminc 0.20. In addition, ampicillin and chloramphenicol were added to IO0 &ml. [ring 2.“C]LHiatidinc was added at 40 &ml, which was sufficient to support groath to n final optical density af 4.5 at 600 nm. CAT production was highly variable; bctwccn 30 and 140 rng protein per litrc of culture. Growth was initiated by inoculation of 500 ml of medium in a 2 litre bnfflcd flask with a starter culture of IO ml and grown overnight at 37’C with continuous shaking. Cells were then harvested and CAT purified as described previously [I 11, For NMR analysis CAT was transferred into 50 mM N~zHPO~//VUI-I~PO~ in 99.9% D,O by SUC~CS~~VC concentration and dilution over an Amicon YMlO inem. brane. All NMR spectra wcrc obtained on a Brukcr AM500 spectrometer at 3 l3K unless stated otherwise. An ‘inverse’ probe was used to obtain all the spectra. Typical pulse lengths were: 90°(‘H)= 12 ps, 90°(“C)= 10 us, 9O”(“C for low power decoupling)=70 ps. Lowpower X-nucleus decoupling was afforded using a Bruker SW BFX-S linear amplifier, The pulse sequences used were chosen for their simplicity, thereby minimizing the loss of magnetization due to relaxation during the pulse sequence, The ID heteronuclear multiple quantum pulse sequence is: 90°(‘H)-l/2J-90°(X)Ia0”(‘H)900(X)-l/2& acq.-, and for the 2D heteronuclear multiple quantum experiment: 90o(‘M)-l/2J-900(X)-t,/2-1800(iH)-t~/2-9Oo(’H)-l/2J-acq., both sequences having phase cycling adapted from [l2]. Solvent suppression in both the 1D and 2D experiments was afforded by using a lowpower pre-irradiation pulse during the relaxation period. Either the CARP 1131 or the WALTZ-16 &coupling sequence was used for Xnucleus decoupling. Typically, for ID datasets, 500 scans were obtained; for 2D datasets, 2K x 256 data matrices were accumulated, with 196 scans per tI value.
125
3. RESULTS AND DISCUSSION Previous attempts to identify the histidine C2-‘H resonances directly in ehe ‘H PJMR spectrum of CATIII met with only very limited success [14], The results in Fig. 1 clearly demonstrate the utility of the labelling procedure snd the heteronuclear ‘3C-‘H multiple quancum coherence method (HMQC) in selecting the histidine C2-‘M signals. Five resonances are readily identified in the HMQC 13Cedited spectrum (Fig. Ib), out of a total of seven histidines in the translated nucleotide sequence of CATIII [9]. The twodimensional heteronuclear correlation r;pcctrum of the sample further separated the histidine C2-‘H signals in the ‘%Z dimension, allowing six resonances to be distinguished (Fig. 2). The signal at Fl = 137.4 ppm and F2 = 4.60 ppm is broad and can only be observed at very low contour levels, or in 1D spectra at a lower pH* (Fig. 3). If the 2D experiment is carried out in NH4Cl under the same conditions as Fig. I, the signal is much sharper. Examination of the sample at a lower pH* revealed that three of the signals had shifted (Fig. 2b). Complete titration curves for a range of p-H* values from 6.0 to 8.0 were obtained using a series of one dimensional spectra (Fig. 3). A series of experiments were set up to attempt to assign H195, the active site histidine, to one 126
of these. Addition of a small excess of chloramphcnicol wer the concentration of CAT subunits did not alter the ‘H or ‘% resonances of any of the signals (Fig. 2~). However, addition of a five fold excess of chloramphenicol did cause one resonance to change its ‘W and 13Cchemical shifts (Fig. 2d- the sharp signal at 3.87 and 138 ppm is from chloramphenicol). Assuming a dissociation constant for chloramphenicol of 3.7 PM [14], the active site of the enzyme should be greater than 90% saturated under both conditions. It is likely, therefore, that the effect of chloramphenicol at a higher concentration is caused by binding to a secondary lower affinity site. There is some evidence to support the existence of such a site from a crystallographic analysis of the binding of P-iodochloramphenicol [3]. Chemical modification of the “C-1abelled enzyme was carried out using 3-(bromoacetyl)chloramphenicol, which specifically alkylates H I95 at the N3 position on the imidazole ring [IS], Subsequent incubation of the enzyme under mild alkaline conditions leads to hydrolysis of the oxy-ester and removal of the chloramphenicol moiety, to yield the N3 (H195) carboxymethylated derivative of CAT. Examination of the spectra of the modified enzyme at Iow and high pH* revealed that there were no significant changes in the ‘H and 13Cchemical shifts, although the two broad signals at 4.55 and 4.35 ppm were much better resolved (Fig. 2e
135 13
c
-139
--
-135 I
t I
t
I
13
1
c
0
(PPm) -137
I
t
t 1 ’
8 ,
I
t
4-39
I
f
b
13 c (PPl-rr)
Fig, 2, 2D HMQC spectra of [ring 2-‘Vjhistidine-labelled CAT (25 mg/ml) in 50 mM Na2HPO4/NaHzPOs recorded at 500 MHZ and 313 IL (a) pW = 7.50; (b) PI-I* =6,24; (c) plus 1.3 mM chloramphenicol (pi-I* = 7.50); (d) plus 5.0 mM chloramphenicol (pH* = 7.50); (e) WI (H195) carboxymethylated derivative (pH* = 7.49); (f) N3 (H195) carboxymethylated derivative (pH * = 6.46). Dioxane was used as a reference in both Fl (67.4 ppm) and F2 (0 ppm).
Volum¢ 21~0. number I
VEILS I,~TTEILK
5.0
M~trch 19~Jl
HMQC ,~pe~:tra were idenlical in I~l~O anti D=O. Nonetheless, it is clear lhal six of the hi~tidine C2.1H resonances in CAT can be clearly identified. Specific isotope labellintt a~d multiple quantum coherence techniqu¢~ can therefore be u~ed to identif.~,~ individual tH NMR signals in ~1protein of overall molecular mass 75 000.
4.5 Aeknowledgem¢/ll¢: We are l~ralcful to the S¢ieuee at~d I-~:ngineerhq! Retear~:l~ Council, the ,Medltsal R¢,teareh Cour~¢il and the Wg'll¢onl¢ l'ru~t for financial ~upporl, and to t)r~ R, ¢,;i~1~a n d J, Feen~y for the Ilift ~a( [ring 2-~k:lt,oht,,tktitt¢.
Chemical Shift
(r~pm) REFERENCES
4.0
Ill Shaw, W.V. ~o.=~,_..--e.
and Idm~w~ky, J
(1%1~) J, llaeteri,~l, 95,
1976-19711, 121 Leslie, A,G.W.. Moody, P.C.E. and Shaw. W.V. (191~81 Pro¢. Nail, Aead, S¢i. U.S.A, 85. 4133=41.17 [.1] l.eslic, A.G,W, (1990) J. Mol. Biol, 213, 167-186,
............
[4] LeMaster, D.M, and Riehards, F.M. (1985) Biodtemi~try 24, "/262-'/268.
[5] Grlffey. R,H. and Redfidd, A,G (19117)QI, Rev, BiopP,,ys. 19, .~1-82.
3.5 6.5
I
~
I
7.0
7.5
8.0
pH Fig, 3, pH* titration of the histidine C2 protons in CAT taken from successive I D HMQC spectra under the same conditions as Fig, 2. Precipitation precluded analysis below pH = 6.0. The signals at 4.55 ppm and 4,35 ppm were not detectable at low pH*.
and f). The titration behaviour was identical with that of the unmodified enzyme. These results indicate that the C2-tH resonance of H195 is not among those observed, presumably because it is too broad to be detected. This could arise from exchange broadening, perhaps due to slow exchange between protonated and unprotonated imidazole [16]. Exchange of the C2JH with solvent was ruled out as a possible cause; the ID
128
[6] Ilax, A,, Ikura, M.. Kay. L,E,, Torchia, D,A, and T~¢hudin. R (1990) J, Magn. Re,on, 86, 304-318. [71 Norwood, T.J.. Boyd, J., Herita~,e, J,E,, Sot're, N and Campbell, I.D, (1990} J. M:=gn. Resort, 87, 488-501. {8] Henderson, P,J.F,, Giddens, R,A. and Jones.Mortimer, M.C, (199./) Biochem. J, 162, 309-320. [9: Murray, I.A., Hawkins, A,R,. Keyte, J,W. and Shaw. W,V. (1988) Biochen'~, J, 252, 173-179. (10] Maniatis, T,. Fritsch, E,F. and Sambrook J. (1982) Molecular Cloning; a Laboratory Manual, Cold Spring Harbor Laboratory, (III Lewendon, A,, Murray, l.A,, Kleanthous, C., Cullis, P.M. and Shaw, W,V, 0988) fliocheatlstry 27, 7385-7390, [12l Ce.vat~agh, J. and Keeler J. 0988) J. Magn, Reson, 77,356-362, [13] Shaka, A,J., Barker. P.B. and Freeman, R, (1985) J. Magn. Resort, 64, 547-552. [14} Kleanthous. C. and Shaw. W.V. (1984) Bochem J. 223, 211-220. (151 Kleanthous. C., Cullis, P.M. attd Shaw. W.V. (1985) Biochemistry 24, 5307-5313. [16] Jarderzky, O, and Roberts, Ci.C.K, (1981) NMR in Molecular Biology, Academic Press, New York.
acctyltransfcraxe (CAT; EC Chloramphcnicol 2.3.1.28) is a homotrimer which catalyses the acccylCo&dependent O-acetylation and inactivation of chloramphcnicol, a modification which confers resistance to the antibiotic on host cells [l]. From the three-dimensional X-ray crystal structure of the type III variant of CAT (CATIII), it is known that N3 of the imidazolc ring of H 195 is suitably placed to act as the proposed base catalyst in the reaction [2,3]. Study of the histidinc C2-‘H rcsonanccs in CAT is complicated by its overall molecular mass incorporation in combination with single and multiple quantum coherence experiments has been particularly valuable in the simplification of ‘H spectra of smaller proteins by [4-T]. We sow here that these methods permit detection of histidine C2-‘H resonances even in proteins as large as CAT. 2. EXPERIMENTAL [ri,fg2-“C’JL-Mistidine at 92% “C was obtained from Fluorochem (Old Glossop, Derbyshire, UK) and a 62% “C from Drs R. Gigg and J. Feeney (NIMR, Mill Hill, London). An E, coli histidine auxotroph (strainJMl100 from Dr R.A. Cooper [S]) was used to selectively incorporate labclled histidine into CAT. Transformation with the
Correspondence address: J.P. Derrick, Department t.hiVerSi~y
Gf
Abbrevlalions: heteronuclear
Published
Lcicestcr, CAT, multiple
of Biochemistry,
Leicester LE? 9!?Hi 1JK chloramphenicol acetyltransferase; quantum coherence
by Elsevier Science Publishers
B. V.
HMQC,
in 6. coii 191, wzu eonduclcd us/& i\ CpCl~ ~ro&iure [LO). Optimai growth conditions for production of CATIII were round to be in n medium containing (in g/l), Nn#PQ.t 6.8, KHzP& 3.0, NaCI 0.5, NH& 1.0, MyS& lI.49, thymine 0. I, glycerol 4.0, I.-arpininc, 0,28, I--uspnraric acid 0.74, L-cystcineO.20, L.glycirrr 0.20, L-glurnmicacid 2.8, L-isolcucinc 0S54, L-l~ucine 0.70, L-lyxine 0.74, L-mcthioninc 0.20, L.~p~ieriylnlatii~ic O.LJ, L.nrolinc 0.80. L-rhrconinc 0.60. Ltryptophjn 0[26, L-ryrosine’0.62, L-valinc d-82, L-&nine 0.3j, LJerinc 0.20, I,-asparnginc 0,23, L&utaminc 0.20. In addition, ampicillin and chloramphenicol were added to IO0 &ml. [ring 2.“C]LHiatidinc was added at 40 &ml, which was sufficient to support groath to n final optical density af 4.5 at 600 nm. CAT production was highly variable; bctwccn 30 and 140 rng protein per litrc of culture. Growth was initiated by inoculation of 500 ml of medium in a 2 litre bnfflcd flask with a starter culture of IO ml and grown overnight at 37’C with continuous shaking. Cells were then harvested and CAT purified as described previously [I 11, For NMR analysis CAT was transferred into 50 mM N~zHPO~//VUI-I~PO~ in 99.9% D,O by SUC~CS~~VC concentration and dilution over an Amicon YMlO inem. brane. All NMR spectra wcrc obtained on a Brukcr AM500 spectrometer at 3 l3K unless stated otherwise. An ‘inverse’ probe was used to obtain all the spectra. Typical pulse lengths were: 90°(‘H)= 12 ps, 90°(“C)= 10 us, 9O”(“C for low power decoupling)=70 ps. Lowpower X-nucleus decoupling was afforded using a Bruker SW BFX-S linear amplifier, The pulse sequences used were chosen for their simplicity, thereby minimizing the loss of magnetization due to relaxation during the pulse sequence, The ID heteronuclear multiple quantum pulse sequence is: 90°(‘H)-l/2J-90°(X)Ia0”(‘H)900(X)-l/2& acq.-, and for the 2D heteronuclear multiple quantum experiment: 90o(‘M)-l/2J-900(X)-t,/2-1800(iH)-t~/2-9Oo(’H)-l/2J-acq., both sequences having phase cycling adapted from [l2]. Solvent suppression in both the 1D and 2D experiments was afforded by using a lowpower pre-irradiation pulse during the relaxation period. Either the CARP 1131 or the WALTZ-16 &coupling sequence was used for Xnucleus decoupling. Typically, for ID datasets, 500 scans were obtained; for 2D datasets, 2K x 256 data matrices were accumulated, with 196 scans per tI value.
125
3. RESULTS AND DISCUSSION Previous attempts to identify the histidine C2-‘H resonances directly in ehe ‘H PJMR spectrum of CATIII met with only very limited success [14], The results in Fig. 1 clearly demonstrate the utility of the labelling procedure snd the heteronuclear ‘3C-‘H multiple quancum coherence method (HMQC) in selecting the histidine C2-‘M signals. Five resonances are readily identified in the HMQC 13Cedited spectrum (Fig. Ib), out of a total of seven histidines in the translated nucleotide sequence of CATIII [9]. The twodimensional heteronuclear correlation r;pcctrum of the sample further separated the histidine C2-‘H signals in the ‘%Z dimension, allowing six resonances to be distinguished (Fig. 2). The signal at Fl = 137.4 ppm and F2 = 4.60 ppm is broad and can only be observed at very low contour levels, or in 1D spectra at a lower pH* (Fig. 3). If the 2D experiment is carried out in NH4Cl under the same conditions as Fig. I, the signal is much sharper. Examination of the sample at a lower pH* revealed that three of the signals had shifted (Fig. 2b). Complete titration curves for a range of p-H* values from 6.0 to 8.0 were obtained using a series of one dimensional spectra (Fig. 3). A series of experiments were set up to attempt to assign H195, the active site histidine, to one 126
of these. Addition of a small excess of chloramphcnicol wer the concentration of CAT subunits did not alter the ‘H or ‘% resonances of any of the signals (Fig. 2~). However, addition of a five fold excess of chloramphenicol did cause one resonance to change its ‘W and 13Cchemical shifts (Fig. 2d- the sharp signal at 3.87 and 138 ppm is from chloramphenicol). Assuming a dissociation constant for chloramphenicol of 3.7 PM [14], the active site of the enzyme should be greater than 90% saturated under both conditions. It is likely, therefore, that the effect of chloramphenicol at a higher concentration is caused by binding to a secondary lower affinity site. There is some evidence to support the existence of such a site from a crystallographic analysis of the binding of P-iodochloramphenicol [3]. Chemical modification of the “C-1abelled enzyme was carried out using 3-(bromoacetyl)chloramphenicol, which specifically alkylates H I95 at the N3 position on the imidazole ring [IS], Subsequent incubation of the enzyme under mild alkaline conditions leads to hydrolysis of the oxy-ester and removal of the chloramphenicol moiety, to yield the N3 (H195) carboxymethylated derivative of CAT. Examination of the spectra of the modified enzyme at Iow and high pH* revealed that there were no significant changes in the ‘H and 13Cchemical shifts, although the two broad signals at 4.55 and 4.35 ppm were much better resolved (Fig. 2e
135 13
c
-139
--
-135 I
t I
t
I
13
1
c
0
(PPm) -137
I
t
t 1 ’
8 ,
I
t
4-39
I
f
b
13 c (PPl-rr)
Fig, 2, 2D HMQC spectra of [ring 2-‘Vjhistidine-labelled CAT (25 mg/ml) in 50 mM Na2HPO4/NaHzPOs recorded at 500 MHZ and 313 IL (a) pW = 7.50; (b) PI-I* =6,24; (c) plus 1.3 mM chloramphenicol (pi-I* = 7.50); (d) plus 5.0 mM chloramphenicol (pH* = 7.50); (e) WI (H195) carboxymethylated derivative (pH* = 7.49); (f) N3 (H195) carboxymethylated derivative (pH * = 6.46). Dioxane was used as a reference in both Fl (67.4 ppm) and F2 (0 ppm).
Volum¢ 21~0. number I
VEILS I,~TTEILK
5.0
M~trch 19~Jl
HMQC ,~pe~:tra were idenlical in I~l~O anti D=O. Nonetheless, it is clear lhal six of the hi~tidine C2.1H resonances in CAT can be clearly identified. Specific isotope labellintt a~d multiple quantum coherence techniqu¢~ can therefore be u~ed to identif.~,~ individual tH NMR signals in ~1protein of overall molecular mass 75 000.
4.5 Aeknowledgem¢/ll¢: We are l~ralcful to the S¢ieuee at~d I-~:ngineerhq! Retear~:l~ Council, the ,Medltsal R¢,teareh Cour~¢il and the Wg'll¢onl¢ l'ru~t for financial ~upporl, and to t)r~ R, ¢,;i~1~a n d J, Feen~y for the Ilift ~a( [ring 2-~k:lt,oht,,tktitt¢.
Chemical Shift
(r~pm) REFERENCES
4.0
Ill Shaw, W.V. ~o.=~,_..--e.
and Idm~w~ky, J
(1%1~) J, llaeteri,~l, 95,
1976-19711, 121 Leslie, A,G.W.. Moody, P.C.E. and Shaw. W.V. (191~81 Pro¢. Nail, Aead, S¢i. U.S.A, 85. 4133=41.17 [.1] l.eslic, A.G,W, (1990) J. Mol. Biol, 213, 167-186,
............
[4] LeMaster, D.M, and Riehards, F.M. (1985) Biodtemi~try 24, "/262-'/268.
[5] Grlffey. R,H. and Redfidd, A,G (19117)QI, Rev, BiopP,,ys. 19, .~1-82.
3.5 6.5
I
~
I
7.0
7.5
8.0
pH Fig, 3, pH* titration of the histidine C2 protons in CAT taken from successive I D HMQC spectra under the same conditions as Fig, 2. Precipitation precluded analysis below pH = 6.0. The signals at 4.55 ppm and 4,35 ppm were not detectable at low pH*.
and f). The titration behaviour was identical with that of the unmodified enzyme. These results indicate that the C2-tH resonance of H195 is not among those observed, presumably because it is too broad to be detected. This could arise from exchange broadening, perhaps due to slow exchange between protonated and unprotonated imidazole [16]. Exchange of the C2JH with solvent was ruled out as a possible cause; the ID
128
[6] Ilax, A,, Ikura, M.. Kay. L,E,, Torchia, D,A, and T~¢hudin. R (1990) J, Magn. Re,on, 86, 304-318. [71 Norwood, T.J.. Boyd, J., Herita~,e, J,E,, Sot're, N and Campbell, I.D, (1990} J. M:=gn. Resort, 87, 488-501. {8] Henderson, P,J.F,, Giddens, R,A. and Jones.Mortimer, M.C, (199./) Biochem. J, 162, 309-320. [9: Murray, I.A., Hawkins, A,R,. Keyte, J,W. and Shaw. W,V. (1988) Biochen'~, J, 252, 173-179. (10] Maniatis, T,. Fritsch, E,F. and Sambrook J. (1982) Molecular Cloning; a Laboratory Manual, Cold Spring Harbor Laboratory, (III Lewendon, A,, Murray, l.A,, Kleanthous, C., Cullis, P.M. and Shaw, W,V, 0988) fliocheatlstry 27, 7385-7390, [12l Ce.vat~agh, J. and Keeler J. 0988) J. Magn, Reson, 77,356-362, [13] Shaka, A,J., Barker. P.B. and Freeman, R, (1985) J. Magn. Resort, 64, 547-552. [14} Kleanthous. C. and Shaw. W.V. (1984) Bochem J. 223, 211-220. (151 Kleanthous. C., Cullis, P.M. attd Shaw. W.V. (1985) Biochemistry 24, 5307-5313. [16] Jarderzky, O, and Roberts, Ci.C.K, (1981) NMR in Molecular Biology, Academic Press, New York.
