Chloramphenicol binding site of an fi− R-factor-specified variant of chloramphenicol acetyltransferase

ARCHIVES OF BIOCHEMISTRYAND BIOPHYSICS Vol. 201. No. 1, April 15, pp. 115-120, 1980

Chloramphenicol

YESHAYAHU

Binding Site of an fi- R-Factor-Specified of Chloramphenicol Acetyltransferasel NITZAN (ZAIDENZAIG)

Department

qf Life Sciences,

Bar-Ilatc

AND

Variant

SHOSHANA GOZHANSKY

Unzversity.

Ramat-Cm,

Iwael

Accepted October 30, 1979 Chloramphenicol acetyltransferase (EC 2.3.1.28) specified by the fi- B-factor (type 11) is highly sensitive to sulfhydryl reagents. When this variant was treated with stoichiometric amounts of 2,2’dithiobispyridine, 90% of the enzymatic activity was lost with concomitant introduction of 0.9 to 1.0 thiopyridine groups per mole of enzyme prot orner. In the presence of stoichiometric amounts of the substrate, chloramphenicol, the enzyme was neither inactivated nor modified by the sulfhydryl reagents. .4cetyl-coenzyme A exerted no protective effects when present in the reaction mixture. The enzyme was also inactivated by cyanylation with a stoichiometric amount of 2-nitro-5-thiocyanobenzoic acid. Labeling native type II enzyme with iodo[Y~]acetamide and subsequently subjecting it to peptic digestion yielded one radioactive peptide. This cysteine-containing peptitle had the same sequence as that found near the cysteine close to the chloramphenicol binding site of the commonly occurring type 1 enzyme. In conclusion, this cysteine residue is essential for the catalytic activity of both types of enzyme and is located in or near the chloramphenicol binding site. It also seems that the cysteine in type II is more sensitive to sulfhydryl reagents than the homologous cysteine in type I, probably because it is more available for modification.

Chioramphenicol acetyltransferases (EC 2.3.1.28) were isolated from various bacteria resistant to the antibiotic chloramphenicol(1,2). Variants of chloramphenico1 acetyltransferase can easily be purified to homogeneity by affinity chromatography and in each case consist of tetramers of four identical subunits (molecular weight, 22,500 or 24,500) (3, 4). The enzyme catalyzes the 0-acetylation of chloramphenicol by acetylcoenzyme A (5, 6). In Escherichia coli and related Gram-negative bacteria, three different types of chloramphenicol acetyltransferase have been characterized. Each type or variant is specified by an extrachromosomal element (R-factor) carrying a determinant for chloramphenicol resistance. Significant differences have been observed between the variants with respect to net charge, substrate affinity, immunological reactivity, and sensitivity to sulfhydryl reagents (7). More recent studies have also

revealed differences in elution behavior and in affinity and hydrophobic chromatography (3, 4) and have confirmed and further extended the classification scheme proposed. (2). Chloramphenicol resistance of Gramnegative bacteria is commonly a marker of fi+ (F fertility-inhibiting) R-factors and is also associated with fi- (F fertility-noninhibiting) R-factors. One of the naturally occurring variants of chloramphenicol acetyltransferase (type II), specified by a fiR-factor (designated Sa), has been shown to be particularly sensitive to inhibition by DTNB (7).’ Early studies on chloramphenicol acetylation proposed that essential sulfhydryl groups were probably involved in the mechanism of catalysis (6-8). Recent studies describe the role of cysteine residues in the mechanism of the most common, naturally occurring variant (type I), mediated by fi’ R-factor. It was shown that two

’ A preliminary report of this work was made at the Society for Biochemistry in Jerusalem in April 1979.

? Abbreviations used: DTP, 2,2’-dithiobispyridine; DTNB, 5,5’-dithiobis(2-nitrobenzoic acid); NTCB, 2-nitro-5.thiocyanobenzoic acid.

Israeli

115

0003-9861180/05011506$02.00/0 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

116

NITZAN

AND GOZHANSKY

cysteines were important for enzymatic activity (9). Both cysteines are protected by the substrate chloramphenicol. However, it is probable that one cysteine is located in the vicinity of the chloramphenico1binding site while the other is near the acetyl-CoA site. Although type II variant is mediated by fi- plasmid it has some properties similar to those of the type I enzyme; the most striking difference between the variants is the high sensitivity type II variant exhibits to specific sulfhydryl reagents (2, 7). The present study examines the nature of the chloramphenicol binding site and the reactive cysteine located there. MATERIALS

AND METHODS

Strain and growth conditions. In this study we employed an E. coli strain 553 derived from E. coli K12 carrying an fi- plasmid (R-factor) designated Sa which mediated the constitutive synthesis of chloramphenicol acetyltransferase type II (3, 7). The bacterial strain was kindly provided by Dr. W. V. Shaw. Antibiotic resistance markers were Cma, Stra, Kana, and SUP. The minimal inhibitory concentration for chloramphenicol was greater than 1000 ygiml. Bacterial cells were grown to stationary phase at 37°C in Difco nutrient broth + 50 pgiml chloramphenicol supplemented with glycerol (final concentration, 1% (v/v)). A packed cell paste was obtained after highspeed centrifugation in a Sharples supercentrifuge. The cell paste was resuspended in 50 mM Tris. HCl buffer, pH 7.8 containing 0.05 M 2-mercaptoethanol and the cells were broken by extrusion in a French pressure unit. Crude cell-free extract was obtained by centrifugation at 20,OOOgfor 20 min. Enzyme puri$cation. Chloramphenitol acetyltransferase was precipitated by ammonium sulfate at a final concentration equal to 50% saturation. The protein precipitate was placed in an ice bath for 30 min before being centrifuged at 30,OOOg for 20 min. The precipitate was dissolved in 50 mM Tris’ HCl buffer, pH 7.8, dialyzed, and purified by affinity chromatography (3). In outline form, purification was achieved by applying the dialyzed extract on a substituted chloramphenicol base affinity resin. Chloramphenicol acetyltransferase type II was eluted by a solution of 0.6 M sodium chloride and 5 mM chloramphenicol in 50 mM Tris’ HCl, pH 7.8. Enzyme activity and spectrometric measurements. Chloramphenicol acetyltransferase activity was assayed spectrophotometrically using a Gilford spectrophotometer (10). Since the enzyme is sensitive to DTNB (7) and since chloramphenicol protects against inactivation by DTNB, the procedure was

modified. The reaction mixture in each l-ml cuvette was composed of 0.1 M Tris HCl, pH 7.8, 1 mM DTNB, 0.25 mM chloramphenicol, and 5 to 10 ~1 of enzyme preparations. The reaction was started by the addition of acetyl-CoA (final concentration, 0.1 mM). The concentration of Z-nitro-5-thiobenzoate produced was measured by absorbance at 412 nm (E = 13.6 x 10” M-' cm-‘) and the concentration of 2-thiopyridine by absorbance at 343 nm (e = 8.08 x lo3 M-I cm-') (11). Amino acid analysis and sequence. Amino acid compositions of type II radioactively labeled peptides were analyzed by the procedure of Moore and Stein (12) using norleucine as an internal standard. Hydrolysis was performed in racuo at 105°C for 24 h. The hydrolyzed samples were analyzed on a TSM Technicon amino acid autoanalyzer. The number of sulfhydryl groups was estimated by reaction with iodo114C]acetamide in 6 M guanidine-HCl. For determination of the amino acid sequence of the radioactively labeled peptides, the dansyl-Edman procedure was used (13). Radioactive peptides were isolated from the peptic digests and purified by high-voltage paper electrophoresis at pH 6.5 (pyridine acetate) followed by electrophoresis at pH 1.9 (acetic-formic acid).

RESULTS

Titrating the thiol groups in the native type II variant with DTP or DTNB results in the introduction of thiopyridine or thionitrobenzoate groups, respectively, into the enzyme molecule. Figure 1 shows that one cysteine per protomer (or four cysteines per tetramer) is modified by the addition of a stoichiometric amount of DTP. A second cysteine is modified only when the reagent at eightfold higher concentrations is reacted with the enzyme. Titration with DTNB requires 3 mol of reagent to introduce 1 mol of thionitrobenzoate group per monomer. A second cysteine residue is modified by 16 mol of DTNB per mole of enzyme protomer (point not shown in Fig. 1). The total number of cysteines per protomer of type II variant was estimated by titrating the denatured form of the enzyme with iodo[14C]acetamide. The native enzyme was denatured in 6 M guanidine-HCl, pH 7.5. We calculated that 4.4 cysteine residues are found in one monomer and estimated that four half-cysteines are associated with 22,500 g of chloramphenicol acetyltransferase type II protomer. Our data so far indicate that one cysteine residue (out of the

REACTIVE

CYSTEINE

OF CHLORAMPHENICOL

four per protomer) is readily modified by the chromogenic disulfide reagents and that it is modified better by the noncharged reagent (DTP) than by the charged one (DTNB). Treatment of native type II chloramphenicol acetyltransferase with a stoichiometric amount of DTP results in a loss of 90 to 95% of the enzyme activity (Fig. 2). It seems that modifying the first cysteine residue by stoichiometric amounts of the reagent, as shown in Fig. 1, leads to complete inactivation. However, if chloramphenicol (one of the enzyme’s substrates) is present in the reaction mixture, the enzyme is protected against inactivation. One mole of chloramphenicol per mole of enzyme protomer is sufficient to protect the enzyme completely against inactivation. Two moles of chloramphenicol are sufficient to protect type II even when it is treated with 10mol of DTP per mole of enzyme protomer. The sensitivity of native type II chloramphenicol acetyltransferase to thiol reactive reagents was confirmed by the data summarized in Table I. The rates of inactivation by the various sulfhydryl reagents were calculated from the equation, In E/E,, = -kt, where E, is the initial enzyme activity and E is the enzyme activity at time t (14). A

REAGENT

CONCENTRATION(,LMI

FIG. 1. Titration of native chloramphenicol acetyltransferase type II with sulfhydryl agents. Thiol groups of chloramphenicol acetyltransferase type II (90,000 M,) were titrated by increasing amounts of 5,5’-dithiobis(2-nitrobenzoic acid) (A) and 2,2’dithiobispyridine (B) which were added at intervals of 10 min. The initial concentration of the enzyme protomer in 0.1 M Tris. HCl, pH 7.8,37”C was 10.5 pM and the final concentration was 9.4 PM. The number of thiol groups that have reacted per subunit (22,500 J4,) or per tetrameric form are plotted against the concentrations of the reagents added.

ACETYLTRANSFERASE

0

2 MOLES ENZYME

4

TYPE

6

REAGENT

El PER

MOLE

II

117

IO OF

PROTOMER

E’IG. 2. Inactivation of chloramphenicol acetyltransferase type II with 2,2’-dithiobispyridine in the absence and presence of chloramphenicol. Chloramphenicol acetyltransferase (0.18 mgiml) was reacted at pH 7.8, 37°C with the indicated amounts of DTP (A) and in the presence of 1 mol of chloramphenico1 per mole of enzyme protomer (B) or 2 mol of chloramphenicol per mole of enzyme protomer (C). After 30 min of reaction under the various conditions enzymatic activity was assayed.

comparison of inactivation rates obtained for type I (9) and type II enzymes shows that type II is far more sensitive to sulfhydry1 reagents than type I. In contrast to the type I chloramphenicol acetyltransferase, the type II enzyme is also sensitive to the negatively charged reagents such as DTNB and iodoacetic acid. Since type I was sensitive only to uncharged reagents it was assumed that it has an aspartic or glutamic residue near the cysteine residue which prevents modification by negatively charged reagents (9). The introduction of the radioactively labeled reagents iodo[14C]acetamide and fluorodinitro[‘4C]benzene showed that chloramphenicol prevents modification of the cysteine residue and subsequent inactivation of the enzyme but acetyl-coenzyme A does not (Table II). These data strongly suggest that the reactive cysteine which is rapidly modified by the thiol reagents is located at the chloramphenicol binding site. Furthermore, the rate of inactivation of the type II enzyme is strongly affected by pH. Assuming this is due to ionization of the functional cysteine group, this cysteine would have an apparent pK of 7.5. A similar apparent pK was found for the functional

118

NITZAN TABLE

AND GOZHANSKY

I

REACTION OFTHIOL-REACTIVEREAGENTSO~VNATIVE

CHLORAMPHE~XOLACETYLTRANSFERASE (TYPE II)"

Reagent Dithiopyridine Dithionitrobenzoic acid Fluorodinitrobenzene Iodoacetamide Iotloacetic acid

Concentration (mM)

Rate of inactivation (K,,,,, x 10”)

0.01

156.8

0.01 0.04 5.0 5.0

34.5 32.2 6.54 21.1

” Native chloramphenicol acetgltransferase type II buffer, pH 7.8 was (0.18 mgiml) in 50 mM Tris.HCl treated with the reagents as indicated. Aliquots were taken at intervals and the loss of activity as a function of time was analyzed. The apparent inactivation rate constants were calculated as described in the text.

cysteine in type I (9). The apparent pK could be followed by the inactivation rates of the enzyme at various pH values in the presence of sulfhydryl reagents (data not shown). The importance of the reactive cysteine residue for enzymatic activity was demonstrated by cyanylation the sulfhydryl group with 2-nitro-5-thiocyanobenzoic acid. This reagent, sterically a large group, modifies the cysteine residue by introducing the small cyanide group (15). This approach is useful in distinguishing between steric interference caused by the ligands present at the modified thiols and participation of the cysteine residue in the catalytic mechanism (16, 17). When chloramphenicol acetyltransferase type II was incubated with a fivefold excess of [14C]NTCB (prepared according to Degani and Patchornik (15)), the enzyme was inactivated 85% and spectrophotometric measurements at 412 nm showed that 0.87 mol of thionitrobenzoate was present per mole of protomer. After dialysis had removed the excess reagent, the modified and inactive enzyme yielded 0.82 mol of [14C]CNp that were introduced per mole of protomer. The lack of activity upon cyanylation further strengthens our assumption that the modified cysteine residue is essential for catalytic activity or that the position of this cysteine at the

active site is so critical that it will tolerate not even substitution by CN-. In order to identify the reactive cysteine and to correlate it to the cysteines known from the type I enzyme, the amino acid sequence around this cysteine was determined. In the method employed, native type II (30 mg in 50 mM Tris. HCl at pH 7.8, 37°C) was treated with iodol14C]acetamide (5 InM) till 90% inactivation was obtained. Under these conditions, 0.95 mol of amino l’“C]carboxymethyl groups was covalently bound to each protomer. The inactivated 14C-labeledprotein was dialyzed overnight at +4”C against 6 liters of 0.01 N HCl and was digested with pepsin (ratio of pepsin to labeled protein, 1:lO by weight). After 8 h at 37°C the digest was applied to a Sephadex G-25 fine column (150 x 1.5 cm). One radioactive peak was eluted by 2% acetic acid. Further purification of the fraction by successive two-step high-voltage TABLE

II

REACTION OF NATIVE CHLORAMPHENICOLACETYLTRANSFERASETYPE II WITH RADIOACTIVE IODOACETAMIDEAND FLUORODINITROBENZENE'~ 'T label bound (moVmol of protomer)

Reagents

1.10

Iodoacetamide alone Iodoacetamide + chloramphenicol Iodoacetamide + acetyl-CoA Fluorodinitrobenzene Fluorodinitrobenzene + chloramphenicol Fluorodinitrobenzene + acetyl-CoA

alone

Residual enzymatic activity (S:) *5

0.10

90

0.92

10

0.93

10

0.15

90

0.86

1.5

0 Iodo[W]acetamide (5 mM) or fluorodinitro[‘“C]benzene (0.04 mM) reacted with samples of chloramphenicol acetyltransferase (type II) (2 mgiml) in 50 mM Tris.HCl, pH 7.8 at 3’7°C. The reaction took place in the absence or presence of the substrates (0.5 mM chloramphenicol, 1.0 mM acetyl-CoA). The reaction was allowed to proceed for 60 min and then assayed for residual enzymatic activity. The radioactivity covalently bound to the enzyme was monitored after stopping the reaction by extensive dialysis against 0.01 N HCl at 4°C.

REACTIVE

CYSTEINE

OF CHLORAMPHENICOL

electrophoresis at pH 6.5 and pH 1.9 yielded one radioactive peptide (Fig. 3). The radioactive peptide was found by amino acid sequencing method (13) to be identical to the cysteine-containing peptide (S,) of the type I enzyme (Table III). We suggest that this cysteine is present at the chloramphenico1binding site of type I and type II chloramphenicol acetyltransferases. DISCUSSION

In our study we attempted to clarify the role of the reactive cysteine residue of type II chloramphenicol acetyltransferase (2, 7, 18). Our experiments focus attention both on the site likely to be involved in chloramphenicol binding (19) and on the presence of a highly reactive cysteine residue at this site. When comparing our data with those for the type I enzyme studied earlier (9) we found that the following properties were exhibited by both variants: (a) Stoichiometric amounts of DTP inactivated the enzyme with concomitant modification of 1 mole of cysteine residue per mole of enzyme

n

4 3 2

B

IL

I

20 DISTANCE

I 111 I

IO

0 FROM

IO ORIGIN

20 (cm,

FIG. 3. High-voltage electrophoresis ofpeptic digest of amido[‘4C]carboxymethyl chloramphenicol acetyltransferase type II. Paper electrophoresis (Whatman 1MM) was performed at pH 6.5 (pyridine acetate) for 120 min at 60 V/cm and a side strip was cut and monitored for radioactivity (A). The radioactive peptide was identified by radioautography (B) and subjected to a second electrophoresis at pH 1.9 (aceticformic acid) for 60 min at 40 V/cm (C). The site of the sample application is indicated by the arrow. The black areas in panels B and C indicate the radioactive peptide. The dotted areas indicate ninhydrin-positive materials.

ACETYLTRANSFERASE TABLE

TYPE

II

119

III

AMINO ACID SEQUENCEOF CHLORAMPHENICOL BINDING SITE OF CHLORAMPHENICOL ACETYLTRANSFERASEVARIANTS Type 1 Type II

Gin-Ser-Val-Ala-Gln-Cys-Thr-Tyr Ser-Val-Ala-Gln-Cys

protomer; (b) the rates of inactivation by various sulfhydryl reagents had the same pK (7.5); (c) the sequence around the rapidly reacting cysteine was the same; (d) in both enzymes chloramphenicol protected the reactive cysteine from inactivation and modification, while acetylcoenzyme A exhibited no such protective features. All the above similarities mav indicate that although the two types differ in the sequences of their terminal amino acids (2) and other properties (2, 3), the conservation of the chloramphenicol binding site may exist and be similar in both enzymes. This study indicates clearly that although types I and II have the same cysteine residue at the chloramphenicol binding site, the cysteine residue in the type II enzyme more readily undergoes modification than does the homologous cysteine in type I. Furthermore, type II is sensitive to negatively charged sulfhydryl reagents such as DTNB or iodoacetate, while type I is not sensitive to these reagents (Table I). For type I an ionized aspartate or glutamate residue immediately adjacent to the susceptible thiol was postulated to explain the inability of negatively charged reagents to affect inhibition at neutral pH (9). It seems likely that the three-dimensional structures of the chloramphenicol binding sites of these two types is not identical, although they share the properties mentioned above. The configuration of the substrate binding site of type II enzyme is the reason that the cysteine residue is more available to nucleophilic attack than the homologous cysteine in type I. Second, this configuration of the site counteracts the effect of the carboxylate residue in the attack by the negatively charged reagents. Since the inhibition rates of negatively charged reagents are 0.22 to 0.3 of those of the uncharged reagents, it may indicate

120

NITZAN

AND GOZHANSKY

that the effect of the carboxylate residue is not completely eliminated in type II. The configurational change may be due to a different amino acid sequence in type II or to a shorter amino acid sequence at the amino terminus of the protomer. According to recent studies (2) both these conjectures are plausible. It should be noted that in type I enzyme 31 residues separate the reactive cysteine from the amino terminal; in type II this distance may be shorter, but this has not yet been confirmed (2). The reactive cysteines of type I and type II appear to be essential for catalytic activity and even relatively mild conformational constraints on this cysteine are not tolerated, not even CN substitution. In previous studies on the type I enzyme (9, 20) it could be shown that the chloramphenicol acetyl transferase reaction favors a random order of substrate addition and is not compatible with a mechanism involving an acetyl-enzyme intermediate (9). Analysis of the type II kinetics and the lack of S-acetyl intermediates (data not shown) may indicate that the cysteine residue in type II, as in type I, is not likely to participate in the mechanism of the enzyme reaction through the formation of a covalent acetyl-enzyme intermediate. When type II enzymes were described (7) they were classified according to their sensitivity to sulfhydryl reagents (18). Since then additional variants from other genera of bacteria have been described and found to be sensitive to sulfhydryl reagents (2). These variants, such as chloramphenicol paraintransferases from Haemophilus jluenxae (al), Clostridium perfringens, and Streptomyces acrimycini (2) are sensitive to sulfhydryl reagents. The Haemophilus parain&enxae enzyme has the same amino terminal sequence as type II. At this stage of our knowledge, we can only assume that these variants have reactive cysteines, probably at the chloramphenicol binding site. In contrast to these variants, no evidence exists for the presence of a reactive cysteine residue in staphylococcal chloramphenicol acetyltransferase variants (22); this was also indicated by the inhibition rates with sulfhydryl reagents (2).

ACKNOWLEDGMENTS This work was supported by a grant from the Bat-Sheva de Rothschild Foundation to Y.N. We thank Mr. J. Ashker for assistance in the highvoltage electrophoresis experiments. REFERENCES 1. NITZAN (ZAIDENZAIG), Y., AND SHAW, W. V. (1977) Isr. J. Med. Sci. 13, 645. 2. ZAIDENZAIG, Y., FITTOPV‘,J. E., PACKMAN, I,. C., APEDSHAW W. V. (1979) Eur. J. Biochem. 100, 609-618. 3. ZAIDENZAIG, Y., AMD SHAW, W. V. (1976) FEBS L&t. 62, 266-271. 4. FITTON, J. E., PACKMAN, L. C., HARDFORD, S., ZAIDENZAIG, Y., AND SHAW, W. V. (1978) Microbiology 19’78 (Schlessinger, D., ed.), pp. 249-252, American Society of Microbiology, Washington, D. C. 5. SHAW, W. V. (1967) J. Biol. Chem. X42,687-693. 6. SUZUKI, Y., AND OKAMOTO, S. (1967) J. Biol.

Chem. 242, 4722-4730. 7. FOSTER, T. J., AND SHAW, W. V. (1973) Antimicrob. Agents Chemother. 3, 99-104. 8. SHAW. W. V. (1975) in Methods in Enzymology (Hash, J. H., ed.), Vol. 43, pp. 737-7.55, Academic Press, New York. Y . . ZAIDENZAIG, Y.. AND SHAW, W. V. (197%

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AND BRODSKY, R.

F. (1968)

Antimicrob. Agents Chemother. 1967,257-263. 11. STUCHBURY, T., SHIPTON, ,M., NORRIS, R., PAUL, J., MALTHOUSE. G., BROCKLEHURST, K., HUBERT, J. A. L., APZDSUSCHITZSKY, H. (1975) Rio&em. J. 151, 417-432. 12. MOORE, S., AND STEIN, W. H. (1963) in Methods in Enzymology (Colawick, S. P., and Kaplan, N. O., eds.), Vol. 6, pp. 819-821, Academic Press, New York. 13. HARTLEY, B. S. (1970)Biochem. J. 119, X05-822. 14. COLMAN. R. F. (1969) Biochemistry 8, 888-898 15. DEGANI. Y., AND PATCHORNIK, A. (1974) Biochemistry 13, l-11. 16. DER TERROSSIAN, E., AND KASSAB. R. (1976)

Eur. J. Biochem. 70, 623-628. 17. BIRCHMEIER. W., WILSON, K. J., AND CHRISTEN. P. (1973) J. Biol. Chem. 248, 1751-1759. 18. GAFFNEY, D. F., FOSTER, T. J., AND SHAW. W. V. (1978) J. Gen. Microbial. 109, 351-358. 19. NITZAN (ZAIDENZAIG). Y. (1979) Iv. J. Med. Sci. Abs. 15, 788. 20. TANAKA, H., IZAKI, K., AND TAKAHASHI, H. (1974) J. Biochem. (7’okyoj 76, 1009-1019. 21. SHAW, W. V., BOUANCHAUD, D. H., AND GOLDSTEIN, F. W. (1978) Antimicrob. Agelds. Chemother. 13, 326-330. 22. FITTON, J. E., AND SAAW, W. V. (1979)Biochem. J. 177, 575-582.