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PROTEINS: Structure, Function, and Genetics 51:442– 452 (2003)

Role of ␤-Lactam Carboxyl Group on Binding of Penicillins and Cephalosporins to Class C ␤-Lactamases Cristina Fenollar-Ferrer,1 Juan Frau,1 Josefa Donoso,1 and Francisco Mun ˜ oz*1 Departament de Quı´mica, Universitat de les Illes Balears, Palma de Mallorca, Spain

1

ABSTRACT Molecular models for the Henry Michaelis complexes of Enterobacter cloacae, a class C ␤-lactamase, with penicillin G and cephalotin have been constructed by using molecular mechanic calculations, based on the AMBER force field, to examine the molecular differentiation mechanisms between cephalosporins and penicillins in ␤-lactamases. Ser318Ala and Thr316Ala mutations in both complexes and Asn346Ala and Thr316Ala/Asn346Ala double mutation in penicillin G complex have also been studied. Results confirm that Thr316, Ser318, and Asn346 play a crucial role in the substrate recognition, via their interactions with one of the oxygens of the antibiotic carboxyl group. Both mutation Ser318Ala and Thr316Ala strongly affect the correct binding of cephalotin to P99, the first mainly by precluding the discriminating salt bridge between carboxyl and serine OH groups, and the second one by the Ser318, Lys315, and Tyr150 spatial rearrangements. On the other hand, Ser318Ala mutation has little effect on penicillin G binding, but the Thr316Ala/Asn346Ala double mutation causes the departure of the antibiotic from the oxyanion hole. Molecular dynamic simulations allow us to interpret the experimental results of some class C and A ␤-lactamases. Proteins 2003; 51:442– 452. © 2003 Wiley-Liss, Inc. Key words: ␤-lactam antibiotics; ␤-lactamase; serine enzymes; molecular modeling structures; mutated structure INTRODUCTION ␤-Lactam antibiotics continue to be the most widely used antimicrobials, even though the development of various bacterial resistance mechanisms has considerably reduced the effectiveness of some over the past few decades. One of the most effective resistance mechanisms involves the production of ␤-lactamase enzymes, which affect the hydrolysis of ␤-lactam antibiotics.1,2 ␤-Lactamases can be classified into four different classes (A–D) according to structure. Class A, C, and D ␤-lactamases are serine enzymes, the serine residue acting as the nucleophile in the hydrolysis reaction. Class A ␤-lactamases are also known as “penicillinases” on account of the ease with which they can hydrolyze penicillins, and class C ␤-lactamases as “cephalosporinases” by virtue of their increased activity against cephalosporins.3,4 The greater or lesser ease with which ␤-lactamases can hydrolyze ©

2003 WILEY-LISS, INC.

cephalosporins or penicillins is directly related to the nature of their active sites.5 A sound knowledge of such active sites and of the role of each amino acid in the process is, therefore, crucial with a view to deciphering the mechanism involved in the molecular recognition of the substrate and its hydrolysis; in addition, it can be highly useful for designing more effective drugs against pathogenic bacteria. A number of theoretical and experimental studies have been performed to establish a general mechanism for enzyme action and to identify the specificities of the different types of enzymes to determine why they respond so differently to substances of a high chemical similarity.5–17 To this end, the amino acids at the active sites of both cephalosporinases and penicillinases have been identified, and their substrate-hydrolysis and substratebinding roles have been elucidated; in addition, the equivalent positions of the amino acids in both ␤-lactamase classes have been determined. Specifically, Ser64 at the active site of the class C ␤-lactamase Enterobacter cloacae P99 has been identified as the nucleophile that attacks the ␤-lactam carbonyl.10 In addition, the amino groups of Ser318 and the previous Ser64 have been found to form hydrogen bonds with the ␤-lactam carbonyl (A and B in Fig. 1),5,7,10,18 as amino groups of Asn152 and Gln120 have with the carbonyl group on the side-chain (G and J in Fig. 1),5,7,10,19 and the carbonyl group in Ser318 with the amino group on the side-chain (F in Fig. 1). 5,7,10,11 Finally, the hydroxyl groups in Tyr150, Ser318, and Thr316 have been found to interact with the ␤-lactam carboxyl, and so have the amino groups in Lys315 and Asn346 (interactions D, H, I, E, and K in Fig. 1).6 – 8,16 –18,20 –23 The relative location of these amino acids is preserved by virtue of the formation of various hydrogen bonds with other amino acid residues at the active site. Such is the case with Lys315 and Ser318; the former is bound by interaction with its amino group and the hydroxyl group in Thr316 and the latter by formation of a strong hydrogen bond between its hydroxyl group and the amino group in Arg349.

Grant sponsor: Spanish government; Grant number: DGICYTBQU 2000-0214. *Correspondence to: Francisco Munõz, Departament de Quı´mica, Universitate de les Illes Balears, Ctra. Valldemossa Km 7.5, E-07071, Palma de Mallorca, Spain. E-mail: [email protected] Received 7 May 2002; Accepted 24 October 2002

RECOGNITION MECHANISM IN ␤-LACTAMASES

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Fig. 1. Interactions between a ␤-lactam (cephalosporin) and the amino acids in the class C ␤-lactamase P99 (bold) and its most common equivalent amino acids in class A ␤-lactamases.

As confirmed by theoretical and experimental studies conducted both by other authors5–7,10,11 and ourselves on the Henry–Michaelis complexes of Staphylococcus aureus PC-116 and Enterobacter cloacae P99,17 and on those of a variety of other ␤-lactamases, effective establishment of the above-described interactions seem to dictate the course of the subsequent enzyme hydrolysis process. One of the most salient conformational differences between penicillins and cephalosporins is the relative orientation of the carboxyl group. In penicillins, the group is bonded to C3 and points to the ␣-side. In cephalosporins, it is bonded to C4, which lies in the same plane as the ␤-lactam ring by virtue of its sp2 hybridization.2 The most immediate interaction between the enzyme and antibiotic is assumed to be that with the carboxyl group; consequently, one can expect the different orientation of this group in both antibiotics to result in different conformations and interactions at the active site. The results of a recent study where we examined the Henry–Michaelis complexes of the ␤-lactamase P99 with various cephalosporins and penicillins appear to confirm this assumption.17 As shown in this work, cephalosporins establish strong interactions D, E, and H, and penicillins D, E, and I but not H (Fig. 1).

Mutagenic studies on the amino acid residues involved in these interactions (viz. Tyr150, Lys315, Thr316, and Ser318) and their analogs in class A ␤-lactamases (viz. Ser130, Lys234, Ser235, and Ala237) (Fig. 1) have revealed marked changes in the hydrolytic activity of penicillins and cephalosporins, particularly on replacement of the residues at positions 316 and 318 in class C ␤-lactamases or their analogs at 235 and 237 (316 of 235 and 318 of 237) in class A ␤-lactamases.18,22,24 –36 Because these residues play no direct role in the hydrolysis mechanism, their significance lies in molecular recognition and in retention of the three-dimensional structure of the active site. In a previous article17 we showed that the carboxyl group of ␤-lactam is of crucial importance for binding and later relative rearrangement of the antibiotic. In this work, we carefully examined the molecular differentiation mechanisms between cephalosporins and penicillins in ␤-lactamases. To this end, the Henry–Michaelis complexes of the class C ␤-lactamase P99 with the antibiotics penicillin G and cephalothin were modeled. The Ser318Ala and Thr316Ala mutations in both complexes and the Asn346Ala and Thr316Ala/Asn346Ala double mutation in that of penicillin G were also studied.

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TABLE I. Interactions Distances (Å) Between the Active Site Amino Acids of ␤-Lactamase P99 and the Antibiotics Cephalothin and Penicillin G in the Different Henry–Michaelis Complexes Modeled

A Ser64[␣NH]–␤-lactam[CO] B Ser318[␣NH]–␤-lactam[CO] C Ser64[␥O]–␤-lactam[CO] D Tyr150[␩OH]–␤-lactam[COO] E Lys315[␩NH3⫹]–␤lactam[COO⫺] F Ser318[␣CO]–␤-lactam[NH side chain] G Asn152[␦NH2]–␤lactam[CO side chain] H Ser318[␥OH]–␤lactam[COO⫺] I Thr316[␤OH]–␤-lactam[COO] J Gln120[␰NH2]–␤lactam[CO side chain] K Asn346[␦NH2]–␤lactam[COO⫺]

Nativecephalothin

Ser318Alacephalothin

Thr316Alacephalothin

Nativepenicillin G

Ser318Alapenicillin G

Thr316Alapenicillin G

Asn346Alapenicillin G

Thr316Ala/ Asn346Alapenicillin G

2.759 1.716 2.836 1.729

2.329 1.703 3.052 1.815

3.508 1.710 3.973 4.058

1.718 1.623 2.654 1.730

1.780 1.635 2.661 1.770

1.639 1.732 2.666 1.851

1.649 1.849 2.645 1.706

1.846 1.751 2.891 4.129

1.670

2.713

6.588

1.662

1.665

3.006

1.650

5.749

1.992

2.185

2.537

2.081

2.248

2.604

1.925

1.734

1.779

4.028

7.217

1.779

1.810

1.762

1.899

5.876

1.724 3.593

— 1.802

1.852 —

3.900 1.694

— 1.703

4.036 —

3.887 1.659

3.249 —

1.744

2.169

10.895

1.785

1.818

1.812

1.801

4.505

5.068

7.896

7.454

1.856

1.781

1.816

—

—

METHODS The crystal structure of the cephalosporinase Enterobacter cloacae P99 was obtained from the Brookhaven Protein Data Bank37 (refcode 2BLT).10 Because of the absence of crystal water molecules, the native structure was supplied with the same water molecules present in the phosphonate derivative of P99 (refcode 1BLS).11 For this purpose, the structure was geometrically optimized stepwise; in this way, the carbon backbone of the enzyme was gradually relaxed by using the protocol of Juteau et al.38 with slight modifications.16,17 Subsequently, each antibiotic was inserted by hand at the active site, and the resulting complex was reoptimized. The charges used in penicillin G and cephalothin were calculated by using Gaussian9439 at the 6 –31⫹G* level. Charges were computed with the aid of the ChelpG system40 normally fitted to the electrostatic potential. The Henry–Michaelis complexes thus obtained were used as the starting conformations for molecular dynamics simulations. Initially, the minimized structures were equilibrated by heating at 300 K for 25 ps using the shake algorithm; this was followed by molecular dynamics simulation for 65 ps (using a timestep of 0.5 ps) and, finally, full optimization of the structures obtained during the simulation. The penicillin G and cephalothin complexes were subjected to various mutations to obtain the following complexes: Thr316Ala–penicillin G, Ser318Ala–penicillin G, Asn346Ala–penicillin G, Thr316Ala/Asn346Ala–penicillin G, Ser318Ala– cephalothin, and Thr316Ala– cephalothin. Following geometric optimization, each conformation was used as the starting point for a molecular dynamics simulation under conditions equivalent to those used with the native cephalothin and penicillin G complexes except for the simulation time, which was extended to 165 ps. All computations were performed on a Silicon Graphics Origin 200 R10000 computer, using the AMBER* force field41,42 as implemented in the Macromodel v. 6.0 software package.43

RESULTS AND DISCUSSION Native Enzyme In the native enzyme, the hydroxyl group in Tyr150 interacts with the amino group in Lys67 (2.363 Å) and Lys315 (2.280 Å). Meanwhile, Arg349 forms two hydrogen bonds, with Asn346 and Ser318 (2.087 and 1.798 Å, respectively). All these distances are similar to those obtained by Lobkovsky et al.10 In addition, in the oxyanion hole solvent molecules solvate some amino acids. Although Lys315 is solvated by W48 and W114 (1.981 and 2.074 Å, respectively), W81 solvates the Ser318 (1.881 Å) and slightly Arg349 (2.576 Å), and W459 solvates the amino group in Asn346 (2.114 Å). Molecular modeling of the complexes of the P99 enzyme with the antibiotics penicillin G and cephalothin provided the values for the most relevant distances between the amino acids of the active site of P99 and ␤-lactam groups shown in Table I. As can be seen, the side-chains in both substrates are bound to the enzyme via virtually identical F, G, and J interactions. However, the H, I, and K interactions of the carboxyl group differ markedly between them. The incorporation of the substrate displaces Lys315, which interacts with the hydroxyl group in Thr316 (1.912 and 2.163 Å in cephalotin-P99 and penicillin G-P99 complexes, respectively). This rearrangement allows Lys315 and Tyr150 to be anchored by O1 of the carboxyl group in the ␤-lactam (E and D). Although in penicillin G complex O2 establishes I and K salt bridges, with the hydroxyl group in Thr316 and the amino group in Asn346, in the cephalotin complex O2 exhibits a single interaction with the hydroxyl group in Ser318 (H). Although O1 interacts similarly in the two antibiotics, the different interactions of O2 influence the orientation of the remainder of the molecule in both substrates.17 The fact that O2 in cephalothin establishes an H interaction results in the ␤-lactam carbonyl forming a single hydrogen bond with Ser318 (B: 1.716 Å) because the Ser64 residue is too distant for this purpose (A: 2.759 Å). In penicillin G, O2 forms two interactions with Thr316 and


Fig. 2. Superposition of active sites in the native–penicillin G and native– cephalothin Henry–Michaelis complexes. The Ser64 residues in each complex are superposed with an RMS ⫽ 0.02 Å, and all atoms other than those in the thiazolidine and dihydrothiazine rings in both antibiotics have been deleted. The amino acid residues Ser64, Thr316, Asn346, and Ser318 in the native–penicillin G complex, and penicillin G itself, are represented as balls and sticks, and those in cephalothin by sticks.

Asn346 (I and K respectively), so the ␤-lactam carbonyl adopts an orientation that allows the formation of two hydrogen bonds of similar strength (A and B). In the native enzyme, the distance between the hydroxyl group in Tyr150 and the amino group in Lys67 is 2.353 Å, which is scarcely modified by the presence of the antibiotic. The salt bridge between O⫺ in Ser64 and the NH3⫹ in Lys67 is strengthened by substrate binding. This distance shortens from 1.888 Å in native enzyme to 1.685 and 1.654 Å in cephalothin and penicillin G complexes. As we stated before, Lys315 is solvated by two water molecules (W48 and W114). The presence of the substrate does not modify this solvation. In the cephalotin-P99 complex, W81 is moved from Ser318 to Arg349 (1.757 Å). On the other hand, in penicillin G-P99 complex, W81 and W459 are scarcely modified. The active sites in the native–penicillin G and native– cephalothin complexes are superimposed in Figure 2 (RMS ⫽ 0.02 Å), which reveals the different spatial arrangement of the amino acids Thr316, Ser318, and Asn346, and carboxyl group of ␤-lactam antibiotic in both complexes. As can be seen, both Thr316 and Asn346 are in favorable positions to interact with O2 in penicillin G complex (I: 1.694 Å and K: 1.856 Å) but not in the cephalothin complex (I: 3.593 Å and K: 5.068 Å). On the other hand, the hydroxyl group in Ser318 is favorably oriented to interact with O2 in the antibiotic in the cephalothin complex (H: 1.724 Å) but too distant from this atom in the penicillin G complex (H: 3.900 Å). Therefore, as found in our previous work,17 positions 318 and 316 play a crucial role in the formation of the complexes; they seem to discriminate between penicillins and cephalosporins via their interactions with one of the oxygen atoms in the ␤-lactam carboxyl.

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The significance of the 316/235 and 318/237 residues has also been exposed in experimental studies of mutagenesis that have revealed marked activity changes in enzymes mutated at such positions. Class A ␤-lactamases usually possess an Ala residue at position 237 and exhibit a high hydrolytic activity on penicillins but a very low one on cephalosporins3,4; simply replacing this amino acid can substantially alter the activity profile of the enzyme. Such is the case with the ␤-lactamases TEM-1 and TEM-10, where the Ala237Thr24 –26 and Ala237Asn24 mutations increase their activity against cephalosporins by up to 380%. They switch from the typical hydrolysis profile of penicillinases to an atypical cephalosporinase profile, usually associated to class C ␤-lactamases. These changes can be explained in the light of the previous assertions as both the Ala237Thr and the Ala237Asn mutations insert a new functional group at this position (a hydroxyl group with the former and an amino group with the latter). This allows the enzyme to establish a new interaction with the substrate, thereby facilitating its binding and hydrolysis. Although most class A ␤-lactamases possess an alanine residue at position 237, some underwent evolutive mutation and exhibit a serine (K-1, CTX-M-4-, Sme-1) or threonine residue (PER-1) at that position as a result. However, these are class A ␤-lactamases, and they exhibit a strong hydrolytic action on cephalosporins.27–30 Some investigators have examined the changes in hydrolytic activity caused by substitution of the Ser237 or Thr237 residue in the ␤-lactamases K-1,27 CTX-M-4,28 Sme-1,30 and PER-1.29 The most marked changes were those resulting from the Ser237Ala or Thr237Ala mutation, which decreased the hydrolytic activity on cephalosporins by a factor up to 4 and, hence, suppressed the cephalosporinase character. Available experimental evidence suggests that the atypical cephalosporinase activity of these class A enzymes arises from the presence of a hydroxyl group at that position. Consequently, the loss of such a group suppresses the atypical activity. On the basis of our results, the presence of the hydroxyl group at position 237 allows ␤-lactamases (K-1, CTX-M-4, Sme-1, and PER-1) to interact with the ␤-lactam carboxyl in cephalosporins similarly to P99 with cephalothin (H interaction). In this way, the enzymes recognize the ␤-lactams as substrates and can hydrolyze them more readily. For this reason, those mutations in the enzymes K-1, CTX-M-4, Sme-1, and PER-1 that result in the loss of the hydroxyl group decrease their cephalosporinase character. To check our hypothesis, we also modeled the Henry– Michaelis complexes of the enzyme, mutated at different key positions, with cephalothin and penicillin G as substrates. Ser318Ala Mutated Enzyme We modeled the Henry–Michaelis complex of P99 and cephalothin with the Ser318Ala mutation and conducted a molecular dynamics simulation on it. Figure 3 shows the trajectories for the C atom in the ␤-lactam carboxyl group, O atom in the hydroxyls of Tyr150 and Thr316, and N

446


Fig. 3. Trajectories for the C atom in the ␤-lactam carboxyl group (pink); the O atoms in the hydroxyl groups of Tyr150 (dark blue) and Thr316 (red); and the N atoms in the amino groups of Lys315 (yellow) and Asn346 (blue) in the molecular dynamics simulation for the Ser318Ala– cephalothin complex.

atom in the amino groups of Lys315 and Asn346. As can be seen from Figure 3, the amino acids Lys315, Tyr150, Thr316, and Asn346 experienced virtually no change in their initial positions, whereas the carboxyl group in cephalothin, and hence the antibiotic, gradually departed from the amino acids with which it interacted. That fact shows a clear tendency to leaving the active site. Subsequent minimization of the conformations obtained in the simulation where the antibiotic was displaced from the active site yielded the Ser318Ala– cephalothin complex, the most relevant distances in which are shown in Table I. The active sites in the native– cephalothin and Ser318Ala– cephalothin complexes have been superimposed [Fig. 4(a– c)]. The substitution of Ser318 with an Ala residue is clearly found to preclude the H interaction, which is the only one possible with O2, thereby causing the ␤-lactam carboxyl to rotate 45° [Fig. 4(a)], the hydrogen bond between Ser318 and Arg349 to be lost (1.86 Å), and the Arg349 and Asn346 residues to be displaced by 3.02 and 2.97 Å, respectively, from their initial positions in the native complex [Fig. 4(b)]. In addition, the Ser318Ala mutation results in rearrangement of the other amino

acids in the ␤-strand.24,31,44,45 In fact, the complex underwent an average rearrangement of the chain by ⬃0.8 Å.* The presence of Ala at position 318 also causes Thr316 and Lys315 to be displaced by 0.76 and 0.99 Å, respectively [Fig. 4(c)]. The displacement in Lys315 suppresses the E interaction with the carboxyl group, the distance changing from 1.670 Å in the native– cephalothin complex to 2.713 Å in the Ser318Ala– cephalothin complex (Table I). The loss of this interaction is countered by the establishment of a new one between the hydroxyl group in Tyr150 and the amino group in Lys315 [1.75 Å, Fig. 4(c)]. Lobkovsky et al.10,11 reported such an interaction in the crystal structure of the enzyme P99 and its rupture upon incorporation of a substrate at the active site. Therefore, its presence under these conditions suggests that the active site of the enzyme clearly evolves to the native structure in the absence of substrate. Therefore, the Ser318Ala mutation suppresses the H interaction with the substrate. This result causes the other amino acids at the active site to adopt their conformations

*Arithmetic mean of the heavy-atom displacement in Thr316 and Lys315 caused by the Ser318Ala mutation


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Fig. 4. Superposition of active sites in the native– cephalothin and Ser318Ala– cephalothin complexes. The Ser64 residues in both complexes are superposed (RMS ⫽ 0.1 Å), and Ser64 and all cephalothin atoms other than those in the six-membered ring have been deleted for easier visualization. The amino acids and cephalothin in the mutated complex are represented by balls and sticks and those belonging to the native– cephalothin complex by sticks. The three parts shown correspond to (a) Thr316 –Gly317–Ser318Ala and the ␤-lactam cephalothin; (b) Ser318Ala–Asn346 –Arg349; and (c) Tyr150 –Lys315–Thr316.

in the native enzyme. This, together with displacement and rotation, causes cephalothin to lose most of its interactions at the active site and hence to depart from it. We also modeled the Ser318Ala–penicillin G complex and used it as the starting point for a molecular dynamics simulation. Figure 5 shows the trajectories for the most relevant amino acids of the active site that fit the carboxyl group of penicillin G and the C atom in the same group. As can be seen, the carboxyl group and amino acids were scarcely altered by the mutation and essentially retained their initial positions.

Optimization of the conformations obtained from the previous simulation yielded the structure for the Ser318Ala–penicillin G complex. The interactions of penicillin within the active site of this complex are shown in Table I. As can be seen, the ␤-lactam carbonyl preserves the A and B hydrogen bonds (1.780 and 1.635 Å, respectively), which are of similar strength to those in the native complex (1.718 and 1.623 Å). The C attack distance continues to be 2.661 Å, and the hydrogen bonds that anchor the side-chain are similarly strong (2.248, 1.810, and 1.818 Å for the F, G, and J, respectively) as in the

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Fig. 5. Trajectories for the C atom in the ␤-lactam carboxyl group (pink); the O atoms in the hydroxyl groups of Tyr150 (dark blue) and Thr316 (red); and the N atoms in the amino groups of Lys315 (yellow) and Asn346 (blue) in the molecular dynamics simulation for the Ser318Ala– penicillin G complex.

native complex. The interactions of the carboxyl group are slightly altered; thus, the K interaction is somewhat stronger (the distance decreases from 1.856 to 1.781 Å). The Ser318Ala mutation preserves roughly all interactions at the active site and even strengthens some, thereby improving binding of the substrate and increasing hydrolytic activity. This is consistent with the experimental results, which show that Ser237Ala (in class A) and Ser318Ala (in class C) mutations increase the hydrolytic activity on penicillins even though they decrease that on cephalosporins.18,24,27,28 Thr316Ala and Asn346Ala Mutated Enzymes Class A ␤-lactamases usually possess a serine residue at position 235, which is the equivalent of 316 in class C ␤-lactamases. Substitution of this serine residue by an alanine residue in TEM-1 slightly decreases its hydrolytic action on penicillins,32–35 similarly to the Thr316Ala mutation in class C ␤-lactamases. In both cases, the mutation causes the loss of a hydroxyl group at 235 (or 316), which precludes an interaction with the substrate at that position. As shown above, the hydroxyl group of Thr316 in the native–penicillin G complex interacts with the O2 atom in the ␤-lactam carboxyl group (I interaction) in penicillins but not in cephalosporins. The loss of the hydroxyl group at position 316 (or 235) by the effect of the Thr316Ala,21 Thr316Val,36 and Ser235Ala32,34,35 mutations prevents the enzyme from establishing an I interaction. On the basis of the above-described results, however, this need not be crucial in either case because the O2 atom in penicillins can still interact with the enzyme via the Asn346 residue (K interaction), whereas that in cephalosporins never interacts in this way (I interaction).

Fig. 6. Trajectories for the C atom in the ␤-lactam carboxyl group (pink); the O atoms in the hydroxyl groups of Tyr150 (dark blue) and Ser318 (green); and the N atoms in the amino groups of Lys315 (yellow) and Asn346 (blue) in the molecular dynamics simulation for the Thr316Ala– penicillin G complex.

We modeled the Henry–Michaelis complex between penicillin G and the enzyme P99 with the Thr316Ala mutation to examine carefully the changes induced by the loss of the hydroxyl group at position 316 (or 235 in class A ␤-lactamases), which seem to decrease the penicillinase character of the enzyme. We also conducted a molecular dynamics simulation of the complex by monitoring its changes with time. Figure 6 shows the trajectories for the C atom in the penicillin G carboxyl group, and the amino acids that fit this group. As can be seen, the amino acids binding the ␤-lactam carboxyl group and the group itself were scarcely displaced from their initial positions. The slight change observed affected all groups as a whole, so relative distances between them remained virtually unchanged. Optimization of the conformations identified during the simulation led to the Thr316Ala–penicillin G complex (see Table I). In this complex, the ␤-lactamase retains A and B hydrogen bonds (1.639 and 1.732 Å, respectively) that are similarly strong to those in the native complex (1.718 and 1.623 Å). The C, G, and J distances are also preserved (2.666, 1.762, and 1.812 Å, respectively), but not the F distance which increases to 2.604 Å. The loss of the hydroxyl group at position 316 precludes binding of the Lys315 residue, which loses its E interaction with the carboxyl group, the distance increasing from 1.662 Å in the native complex to 3.006 Å in the mutated complex. The NH3⫹ in Lys315 remains strongly solvated by two water molecules. However, the favorable orientation of the carboxyl group, which is still bound via D and K interactions (1.851 and 1.816 Å, respectively), preserves the stability of the antibiotic at the enzyme site. Consequently, although


the Thr316Ala mutation causes the loss of the I and E interactions, it introduces no substantial changes in the orientation of the substrate into the active site because the presence of the Asn346 residue allows the establishment of a strong K interaction with the O2 atom in the carboxyl group. The presence of such an interaction suggests that, even though the I interaction is lost, the enzyme can still recognize the penicillin as a substrate so its penicillinase activity is only slightly decreased.32–35 From the foregoing it follows that mutations at position 346, resulting in the loss of this amino group or in shortening of the side-chain, must preclude a K interaction and hence strong enough binding of the penicillin, thereby decreasing enzymatic activity on these substrates. The Asn346 equivalent residue in class A ␤-lactamases has not yet been identified. Lobkovsky et al.10 analyzed the crystal structure of P99 and proposed Arg244 as the analog. In a recent study, we analyzed interactions between various penicillins at the active site of the class A PC-1 ␤-lactamase, and a very strong interaction was also observed between its carboxyl group and the amino group in Arg244, which appears to confirm that both residues are, in fact, equivalent.16 Various mutations in the Arg244 residue in the class A ␤-lactamases SHV-1, SHV-5, and OHIO-146,47 have been studied. The most marked change in hydrolytic activity was found to result from the replacement of Arg244 with a Ser or Cys residue, which decreased the penicillinase activity of the enzymes. In both mutations, replacing Arg with Ser or Cys increased the distance from the hydroxyl or sulfide group to the carboxyl group (to 5 Å),46 which precluded an interaction between the amino acid at position 244 and the carboxyl group (K interaction). This result altered the orientation of the penicillin at the active site and slightly decreased the hydrolytic activity of the enzyme. If the residues at 244 and 346 in the two enzyme classes are assumed to be equivalent, then, by extrapolation, the mutation of Asn346 in a class C ␤-lactamase with any amino acids resulting in the absence of an electrophilic group at this position or in a shortened chain must cause a slight decrease in the activity of these ␤-lactamases on penicillins. Modeling of the Henry–Michaelis complex between penicillin G and P99 with the Asn346Ala mutation, subsequent molecular dynamics simulation, and minimization of the conformations obtained also led to a minimum energy structure (viz. the Asn346Ala–penicillin G complex), the values for the most salient distances in which are listed in Table I. Figure 7 shows the trajectories for amino acids that fit the carboxyl group and for the C atom of this group. As can be seen, the variation was similar to that in the Thr316Ala–penicillin G complex because both the amino acids binding the carboxyl group and the carboxyl group itself were only slightly displaced from their initial positions. As noted earlier, mutations at position 235 (or 316) not only alter hydrolytic action on penicillins but also change the cephalosporinase character of the enzyme. Specifically, the Ser235Ala mutation in class A ␤-lactamases and the

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Fig. 7. Trajectories for the C atom in the ␤-lactam carboxyl group (pink); the O atoms in the hydroxyl groups of Tyr150 (dark blue), Thr316 (red) and Ser318 (green); and the N atom in the amino group of Lys315 (yellow) in the molecular dynamics simulation for the Asn346Ala– penicillin G complex.

Thr316Ala mutation in class C ␤-lactamases decrease their cephalosporinase activity.32,34,35 This finding can not be easily understood in the light of the results initially obtained in the modeling of cephalosporins, based on which interactions of the I type must be insubstantial. We modeled the Henry–Michaelis complex between cephalothin and P99 with its Thr316 residue substituted by an Ala residue and conducted a molecular dynamics simulation on it. Figure 8 shows the trajectories for the C atom in the carboxyl group and for the amino acids that fit this group. As can be seen, the amino acids anchoring the carboxyl group in the ␤-lactam were scarcely displaced from their initial positions; however, the carboxyl group, the rest of the antibiotic molecule, was more markedly displaced and gradually departed from the active site as the simulation progressed. Minimization of the structures obtained in the molecular dynamics simulation where the antibiotic was displaced from the active site in the enzyme yielded the structure of the Thr316Ala– cephalothin complex (Table I). In this complex, the ␤-lactam only retains the B hydrogen bond and the H interaction with the Ser318 (1.710 and 1.852 Å, respectively). The attack distance, 3.793 Å, is roughly 1 Å longer than in the native complex (2.836 Å). The increased C distance, together with the loss of the D and E interactions, and the rupture of the A, F, G, and J hydrogen bonds, indicates that cephalothin is unfavorably arranged at the active site. By superimposing the active sites in the native– cephalothin and Thr316Ala– cephalothin complexes one obtains a clearer picture of the structural changes involved in the process [Fig. 9(a,b)]. The Thr316Ala mutation involves rearrangement of the ␤-strand that contains it. As a result, the Ser318 residue is

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Fig. 8. Trajectories for the C atom in the ␤-lactam carboxyl group (pink); the O atoms in the hydroxyl groups of Tyr150 (dark blue) and Ser318 (green); and the N atoms in the amino groups of Lys315 (yellow) and Asn346 (blue) in the molecular dynamics simulation for the Thr316Ala– cephalothin complex.

displaced by 1.57 Å from its initial position in the native complex. This rearrangement causes the carboxyl group, and hence the remainder of the antibiotic molecule, to rotate 49° and be displaced by 2.33 Å to retain the H interaction [Fig. 9(a)]. This rotation and displacement force the substrate to adopt an unfavorable orientation at the active site and result in the loss of the D interaction (4.058 Å). On the other hand, the removal of the hydroxyl group at 316 prevents the formation of a hydrogen bond with the amino group in Lys315 [1.91 Å Fig. 9(b)]. This results in Lys315 being displaced by 2.41 Å and in the E interaction with the ␤-lactam carboxyl being suppressed (6.588 Å). To counter these changes, a new hydrogen bond with the hydroxyl group in Tyr150 [1.88 Å; Fig. 9(b)] is formed, and the hydroxyl group is displaced by 2 Å as a result. As noted earlier, the outcome of this rearrangement is the departure of the antibiotic from the active site, even though it preserves the H interaction, which renders the complex inactive. Thr316Ala/Asn346Ala Mutated Enzyme

Fig. 9. Superposition of active sites in the native– cephalothin and Thr316Ala– cephalothin complexes. The Ser64 residues in both complexes are superposed (RMS ⫽ 0.06 Å), and all cephalothin atoms other than those in the six-membered ring have been deleted for easier viewing. The amino acids and cephalothin in the mutated complex are represented by balls and sticks and those belonging to the native– cephalothin complex by sticks. The two parts shown correspond to (a) Thr316 –Gly317– Ser318 and the ␤-lactam cephalothin; and (b) Tyr150 –Lys315–Thr316Ala.

The previous results show that, although the Thr316Ala and Asn346Ala mutations suppress the I and K interactions independently, they cause no substantial changes in the Henry–Michaelis complexes or the penicillinase character of the enzyme, and this contradicts the results obtained by altering positions 318 and 237 in cephalosporinases. To analyze the relative significance of the two I and K interactions in the penicillin complexes, the amino acid residues Thr316 and Asn346 were simultaneously replaced with an Ala residue. Figure 10 shows the trajecto-

ries of the molecular dynamics simulation of the Thr316Ala/ Asn346Ala-penicillin G complex. As can be seen, the double mutation caused a slight, simultaneous displacement of the amino acids binding the carboxyl group, as well as the gradual departure of the group from the residues interacting with it. Minimization of the conformations where the antibiotic was displaced from the active site yielded the Thr316Ala/ Asn346Ala–penicillin G complex. The interactions estab-


Fig. 10. Trajectories for the C atom in the ␤-lactam carboxyl group (pink); the O atoms in the hydroxyl groups of Tyr150 (dark blue) and Ser318 (green); and the N atoms in the amino groups of Lys315 (yellow) in the molecular dynamics simulation for the Thr316Ala/Asn346Ala– penicillin G complex.

lished by penicillin G in this new complex are shown in Table I. The gradual departure of the carboxyl group from the amino acids binding it reflected in the loss of the D and E interactions with Tyr150 and Lys315 and in the rupture of the G and J hydrogen bonds with the side-chain (5.876 and 4.505 Å, respectively, Table I). Although the A, B, and F hydrogen bonds were preserved (1.846, 1.751, and 1.734 Å), and the attack distance (2.891 Å) was very similar to that in the native– cephalothin complex (2.836 Å), the loss of the D and E interactions, in addition to the rupture of the G and J hydrogen bonds, involve an adverse orientation within the active site of the enzyme. By superimposing the active sites in the native– penicillin G complex and the doubly mutated complex (Fig. 11), the above-described differences were clearly exposed. The Thr316Ala mutation suppresses the hydrogen bond between the hydroxyl group in Thr316 and the amino group in Lys315 (2.16 Å, Fig. 11). Because this hydrogen bond binds the amino group in Lys315 at a suitable position for interacting with O1 in the ␤-lactam, such a loss displaces the amino acid by 1.43 Å from its initial position and suppresses the E interaction with the antibiotic (5.749 Å, Table I). The loss of three of the four interactions that bind the carboxyl group causes the antibiotic to rotate 40° and be displaced by 2.02 Å from the active site (Fig. 11), thereby suppressing the D interaction (4.129 Å) and resulting in an unfavorable arrangement of the antibiotic at the active site. This is not the case if the Asn residue at position 346 is retained because a K interaction binds the carboxyl group at a suitable position for D and E to be formed (or preserved if they already exist). The poor orientation of the penicillin and the change in its active site toward the native structure confirm the

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Fig. 11. Superposition of active sites in the native–penicillin G and Thr316Ala/Asn346Ala–penicillin G complexes. The Ser64 residues in both complexes are superposed (RMS ⫽ 0.03 Å), and all penicillin G atoms other than those in the five-membered ring have been deleted for easier visualization. The amino acids and penicillin G in the mutated complex are represented by balls and sticks, and those belonging to the native– cephalothin complex by sticks. The figure represents the Asn346Ala–Thr316Ala–Lys315–Tyr150 residues and the ␤-lactam penicillin G.

tendency of the substrate to depart from the active site (Fig. 10). As a result, the Thr316Ala/Asn346Ala double mutation must substantially decrease penicillinase activity in class C ␤-lactamases and by analogy, the Ser235Ala/ Arg244Ala double mutation must have the same effect on class A ␤-lactamases. REFERENCES 1. Fre`re JM. ␤-Lactamases and bacterial resistance to antibiotics. Mol Microbiol 1995;16:385–395. 2. Waley SG. ␤-Lactamase: mechanism of action. In: Page MI., editor. The chemistry of ␤-lactams. Glasgow: Chapman & Hall; 1992. p 198 –228. 3. Ambler RP. ␤-Lactamases and bacterial resistance to antibiotics. Philos Trans R Soc Lond [Biol] 1980;289:321–331. 4. Matagne A, Lamotte-Brasseur J, Fr`re JM. Catalytic properties of class A ␤-lactamases: efficiency and diversity. Biochem J 1998;330: 581–598. 5. Chen CCH, Herzberg O. Structures of the acyl-enzyme complexes of the Staphylococcus aureus ␤-lactamase mutant Glu166Asp: Asn170Gln with benzylpenicillin and cephaloridine. Biochemistry 2001;40:2351–2358. 6. Tsuchida K, Yamaotsu N, Hirono S. Analysis of affinities of penicillins for a class C ␤-lactamase by molecular dynamics simulations. Drug Des Discovery, 1999;16:145–153. 7. Oefner C, D’Arcy A, Daly JJ, Gubernator K, Charnas RL, Heinze I, Hubschwerlen C, Winkler FK. Refined crystal structure of ␤-lactamase from Citrobacter freundii indicates a mechanism for ␤-lactam hydrolysis. Nature 1990;343:284 –288. 8. Patera A, Blaszczak LC, Shoichet BK. Crystal structures of substrate and inhibitor complexes with AmpC ␤-lactamase: pos-

452

9.

10.

11.

12.

13.

14.

15. 16.

17.

18. 19.

20.

21.

22. 23. 24. 25. 26.

27. 28.


sible implications for substrate-assisted catalysis. J Am Chem Soc 2000;122:10504 –10512. Wladkowski BD, Chenoweth SA, Sanders JN, Krauss M, Stevens WJ. Acylation of ␤-lactams by class A ␤-lactamase: an ab initio theoretical study on the effects of the oxy-anion hole. J Am Chem Soc 1997;119:6423– 6431. Lobkovsky E, Moews PC, Liu H, Zhao H, Fre`re JM, Knox JR. Evolution of an enzyme activity: crystallographic structure at 2 Å resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase. Proc Natl Acad Sci USA 1993;90:11257–11261. Lobkovsky E, Billings EM, Moews PC, Rahil J, Pratt RF, Knox JR. Crystallographic structure of a phosphonate derivative of the Enterobacter cloacae P99 cephalosporinase: mechanistic interpretation of a ␤-lactamase transition-state analog. Biochemistry 1994;33:6762– 6772. Dubus A, Normark S, Kania M, Page MGP. The role of tyrosine 150 in catalysis of ␤-lactam hydrolysis by AmpC ␤-lactamase from Escherichia coli investigated by site-directed mutagenesis. Biochemistry 1994;33:8577– 8586. Alvarez-Ibadoy JR, Gonzalez-Jonte R, Hernandez-Laguna A, Smeyers YG. Reaction mechanism of the acyl-enzyme formation in ␤-lactam hydrolysis by means of quantum chemical modeling. J Mol Struct (Theochem) 2000;504:13–28. Pittarch J, Pascual-Ahuir JL, Silla E, Tunõn I. A quantum mechanics/molecular mechanics study of the acylation reaction of TEM-1 ␤-lactamase and penicillanate. J Chem Soc (Perkin 2) 2000;761–767. Zafaralla G, Manavathu EK, Lerner SA, Mobashery S. Elucidation of the role of arginine-224 in the turnover processes of class A ␤-lactamases. Biochemistry 1992;31:3847–3852. Vilanova B, Donoso J, Frau J, Munõz F. Kinetic and molecularmodeling studies of reactions of a class-A ␤-lactamase with compounds bearing a methoxy group on the ␤-lactam ring. Helv Chim Acta 1999;82:1274 –1288. Fenollar-Ferrer C, Frau J, Vilanova B, Donoso J, Munõz F. Molecular modeling studies on Henry-Michaelis complexes of a class C ␤-lactamase and ␤-lactam compounds. J Mol Struct (THEOCHEM) 2002;578:19 –28. Jacobs C, Dubus A, Monnaie D, Normark S, Fre`re JM. Mutation of serine residue 318 in the class C ␤-lactamase of Enterobacter cloacae 908R. FEMS Microbiol Lett 1992;92:95–100. Dubus A, Normark S, Kania M, Page MGP. Role of asparagine 152 in catalysis of ␤-lactam hydrolysis by Escherichia coli AmpC ␤-lactamase studied by site-directed mutagenesis. Biochemistry 1996;34:7757–7764. Monnaie D, Dubus A, Cooke D, Marchand-Brynaert J, Normark S, Fre`re JM. Role of residue Lys315 in the mechanism of action of the Enterobacter cloacae 908R ␤-lactamase. Biochemistry 1994;33: 5193–5201. Dubus A, Wilkin JM, Raquet X, Normark S, Fre`re JM. Catalytic mechanism of active-site serine ␤-lactamases: role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly triad. Biochem J 1994;301:485– 494. Dubus A, Lendent P, Lamotte-Brasseur J, Fre`re JM. The roles of residues Tyr150, Glu272, and His314 in class C ␤-lactamases. Proteins 1996;25:473– 485. Lamotte-Brasseur J, Dubus A, Wade R. pKa calculations for class C ␤-lactamases: the role of Tyr-150. Proteins 2000;40:23–28. Healey WJ, Labgold MR, Richards JH. Substrate specificities in class A ␤-lactamases: preference for penams vs. cephems. The role of residue 237. Proteins 1989;6:275–283. Cantu C, Huang W, Palzkill T. Cephalosporin substrate specificity determinants of TEM-1 ␤-lactamase. J Biol Chem 1997;272:29144 – 29150. Bla´zquez J, Negri MC, Morosini MI, Go´mez-Go´mez JM, Baquero F. A237T as a modulating mutation in naturally occurring extended-spectrum TEM-type ␤-lactamases. Antimicrob Agents Chemother 1998;42:1042–1044. Tamaki M, Nukaga M, Swai T. Replacement of serine 237 in class A ␤-lactamase of Proteus vulgaris modifies its unique substrate specificity. Biochemistry 1994;33:10200 –10206. Gazouli M, Tzelepi E, Sidorenko SV, Tzouvelekis LS. Sequence of the gene encoding a plasmid-mediated cefotaxime-hydrolyzing class A ␤-lactamase (CTX-M-4): involvement of serine 237 in cephalosporin hydrolysis. Antimicrob Agents Chemother 1998;42: 1259 –1262.

29. Bouthors AT, Delettre J, Mugnier P, Jarlier V, Sougakoff W. Site-directed mutagenesis of residues 164, 170, 171, 179, 220, 237 and 242 in PER-1 ␤-lactamase hydrolysing expanded-spectrum cephalosporins. Protein Eng 1999;12:313–318. 30. Sougakoff W, Naas T, Nordmann P, Collatz E, Jarlier V. Role of Ser-237 in the substrate specificity of the carbapenem-hydrolyzing class A ␤-lactamase Sme-1. Biochim Biophys Acta 1999;1433:153– 158. 31. Cantu C, Palzkill T, Huang W. Selection and characterization of amino acid substitutions at residues 237–240 of TEM-1 ␤-lactamase with altered substrate specificity for aztreonam and ceftazidime. J Biol Chem 1996;271:22538 –22545. 32. Imtiaz U, Billings EM, Knox JR, Mobashery S. A structure-based analysis of the inhibition of class A ␤-lactamases by sulbactam. Biochemistry 1994;33:5728 –5738. 33. Rudgers GW, Palzkill T. Identification of residues in ␤-lactamase critical for binding ␤-lactamase inhibitory protein. J Biol Chem 1999;274:6963– 6971. 34. Fonze´ PC, To’th Y. TEM1 ␤-lactamase structure solved by molecular replacement and refined structure of the S235A mutant. Acta Crystallogr 1995;D51:682– 694. 35. Imtiaz U, Manavathu EK, Lerner SA, Mobashery S. Critical hydrogen bonding by serine 235 for cephalosporinase activity of TEM-1 ␤-lactamase. Antimicrob Agents Chemother 1993;37:2438 –2442. 36. Tsukamoto K, Nishida N, Tsuruoka M, Swai T. Function of the conserved triad residues in the class C ␤-lactamase from Citrobacter freundii GN346. FEBS Lett 1990;271:243–346. 37. Bernstein FC, Koetzle TF, Williams GJB, Meyer EF Jr, Brice MD, Rodgers JR, Kennard O, Shimanouchi T, Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol 1997;112:535–542. 38. Juteau JM, Billings E, Knox JR, Levesque RC. Site-saturation mutagenesis and three-dimensional modeling of ROB-1 define a substrate binding role of Ser130 in class A ␤-lactamases. Protein Eng 1992;5:693–701. 39. Frisch MJ, Trucks GW, Schlegel HB, Gill PMW, Johnson BG, Robb MA, Cheeseman JR, Keith T, Peterson GA, Montgomery JA, Raghavachari K, Al-Laham MA, Zakrzewski VG, Ortiz JV, Foresman JB, Cioslowski J, Stefanov BB, Nanayakkara A, Challacombe M, Peng CY, Ayala PY, Chen W, Wong MW, Andres JL, Replogle ES, Gomperts R, Martin RL, Fox DJ, Binkley JS, Defrees DJ, Baker J, Stewart JP, Head-Gordon M, Gonzalez C, Pople JA. Gaussian, Inc., Pittsburgh, PA; 1995. 40. Breneman CM, Wiberg KB. Determining atom-centered monopoles from molecular electrostatic potentials. The need for high sampling density in formamide conformational analysis. J Comp Chem 1990;11:361–373. 41. Weiner SJ, Kollman PA, Nguyen DT, Case DA, Singh UC, Ghio C, Alagona G, Profeta SJ, Weiner P. A new force field for molecular mechanical simulation of nucleic acids and proteins. J Am Chem Soc 1984;106:765–784. 42. Weiner SJ, Kollman PA, Nguyen DT, Case DA. An all atom force field for simulations of proteins and nucleic acids. J Comp Chem 1986;7:230 –252. 43. Mohamadi F, Richards NGJ, Guida WC, Liskamp R, Lipton M, Caulfield C, Chang G, Hend Dickson T, Still WC. MacroModel—an integrated software system for modeling organic and bioorganic molecules using molecular mechanics. J Comp Chem 1990;11:440 – 467. 44. Lamotte-Brasseur Dive G, Dideberg P, Charlier P, Fre`re JM, Ghuysen JM. Mechanism of acyl transfer by the class A serine ␤-lactamase of Streptomyces albus G. Biochem J 1991;279:213–221. 45. Joris B, Dive JM, Renard A, Dideberg O, Charlier P, Fre`re JM, Kelly JA, Boyington JC, Moews PC, Knox JR. The active-siteserine penicillin-recognizing enzymes as members of the Streptomyces R61 DD-peptidase family. Biochem J 1988;250:313–324. 46. Giakkoupi P, Tzelepi E, Legakis NJ, Tzouvelekis LS. Substitution of Arg-244 by Cys or Ser in SHV-1 and SHV-5 ␤-lactamases confers resistance to mechanism-based inhibitors and reduces catalytic efficiency of the enzymes. FEMS Microbiol Lett 1998;160: 49 –54. 47. Lin S, Thomas M, Mark S, Anderson V, Bonomo RA. OHIO-1 ␤-lactamase mutants: the Arg244Ser mutant and resistance to ␤-lactams and ␤-lactamase inhibitors. Biochim Biophys Acta 1999;1432:125–136.