Î²-Lactamases: Why and How - Journal of Medicinal Chemistry (ACS

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β‑Lactamases: Why and How Miniperspective R. F. Pratt* Department of Chemistry, Wesleyan University, Lawn Avenue, Middletown, Connecticut 06459, United States ABSTRACT: The targets of β-lactam antibiotics are bacterial DD-peptidases that catalyze the final steps of peptidoglycan biosynthesis. Bacterial resistance to β-lactams is achieved by the production of β-lactamases, enzymes that catalyze β-lactam hydrolysis. Structural studies of both of these groups of enzymes, their substrates and of β-lactams have led to the conclusion that β-lactamases have evolved from a DDpeptidase ancestor. Thus, the active sites of DD-peptidases and serine β-lactamases are very similar. Why is it then that the active site of a serine β-lactamase can catalyze hydrolysis of a β-lactam while that of a DD-peptidase cannot? In view of the active site similarities, why was it necessary for β-lactamases to evolve at all? The aim of this review is to examine our current understanding of these issues in terms of the crystal structures of the relevant enzymes that are now available, rounding off the analysis with speculation where necessary. he β-lactam antibiotics arguably represent the greatest success story in medicinal chemistry. It is difficult to estimate how many human lives have been saved by these molecules and how many people are now present who would not be had β-lactams not been introduced into medical practice, but the number would be very large. Even now, some 70 years after their introduction, they still represent the most generally used antibiotics.1 Serious bacterial resistance to β-lactams has arisen, of course, particularly through the evolution of βlactamases,2 such that the usefulness of β-lactams in future medicine has been questioned.3 Nonetheless, their targets are still very much valid,4−6 and continued exploration of permutations of their core structure may yet lead to another renaissance.7 The site of action of β-lactam antibiotics has been known since the mid-1960s.8−10 Their targets are the transpeptidase (DD-peptidase) enzymes (aka penicillin-binding proteins, PBPs) that catalyze the final step of bacterial cell wall biosynthesis, the cross-linking of peptidoglycan.11 Resistance to β-lactam antibiotics today is largely due to the presence of βlactamases in most bacteria.12 These enzymes catalyze the hydrolysis of β-lactams and thus negate their antibacterial action. Structural analyses both of the substrates10,13 and of the enzymes14,15 suggested that the serine β-lactamases evolved from DD-peptidases (Scheme 1). Although the major steps in this evolution occurred long ago,16,17 the active sites of these two groups of enzyme remain very similar (Scheme 2).18 It is not immediately clear, therefore, why DD-peptidases cannot catalyze β-lactam hydrolysis and therefore why β-lactamases

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Scheme 2. Arrangement of Conserved Residues in the DDPeptidase/β-Lactamase Active Sitea

a

The nucleophilic serine is Ser1. The vertical line represents the backbone of the β3-strand that forms one side of the active site. All figures assume this orientation.

necessarily had to evolve. This review will address these questions of chemical function largely based on the extensive array of crystal structures of both DD-peptidases and βlactamases now available.12,19,20

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DD-PEPTIDASES As mentioned above, DD-peptidases catalyze the final steps in bacterial cell wall biosynthesis. These steps involve, primarily, the cross-linking (transpeptidase) reaction but also various DDcarboxypeptidase and DD-endopeptidase reactions that are required to produce and process the mature peptidoglycan (Scheme 3). These reactions are catalyzed by a series of DDpeptidases that are found in all bacteria. They have been classified into two groups, high molecular mass (HMM) and low molecular mass (LMM).21 The HMM group is divided into two subgroups, A and B. The former of these contains bifunctional enzymes, catalyzing both the transglycosylase and

Scheme 1

Received: March 25, 2016

© XXXX American Chemical Society

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DOI: 10.1021/acs.jmedchem.6b00448 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Scheme 3. DD-Peptidase Reactionsa

a

The acyl−enzyme intermediate (red), formed by attack of the nucleophilic serine (E-OH) on the substrate, is either hydrolyzed (carboxypeptidase reaction) or aminolyzed (transpeptidase reaction). Cross-linked peptidoglycan can also be recycled to the acyl−enzyme (endopeptidase reaction). The label D indicates the stereochemistry of the chiral centers.

Figure 1. Structural similarities between a β-lactam (benzylpenicillin) and an N-acyl-D-alanyl-D-alanine peptide. The nitrogen of the scissile bond is indicated by an arrowhead in each case. Color code: C, green; H, white; N, blue; O, red; S, yellow.

the transpeptidase (Scheme 3) reactions necessary to incorporate stem peptide monomers into the peptidoglycan polymer. The latter group contains monofunctional transpeptidases. The LMM group, comprising subgroups A, B, and C, are DD-carboxypeptidases and endopeptidases (Scheme 3).19−22 All of these enzymes are efficiently inactivated by βlactams, but antibiotic activity requires inactivation of at least one HMM member. The first question to address is how β-lactams inhibit DDpeptidases and therefore become antibiotics. This question has been discussed, of course, since the nature of the targets became known. The targeted DD-peptidases are serine amidohydrolases (Scheme 3). They react with β-lactams, as with peptide substrates, with initial formation of acyl−enzyme intermediates (Scheme 4), which, in principle, can hydrolyze to

penicillin. Addressing this difference, Lee proposed that penicillin actually more closely resembles the tetrahedral intermediate/transition state of the acylation reaction where the peptide nitrogen would approach a tetrahedral geometry (Figure 1, right, tetrahedral intermediate structures). In view of this comparison, β-lactams can be looked on as transition state analogues, where their ground state geometry allows them to bind to the DD-peptidase active site in an orientation where their intrinsic chemical reactivity is then exploited in facile acylation of the nucleophilic serine of the enzyme active site (EOH, Scheme 4). As has been extensively investigated, the degree of tetrahedrality of the β-lactam nitrogen directly and dramatically affects the chemical reactivity of the β-lactam as an acylation agent. This issue has been extensively explored in the design of penicillins, cephalosporins, and carbapenems by medicinal chemists.23−26 In each of these cases, modulation of reactivity by peripheral substitutions has led to the proliferation of βlactams as antibiotics.27 These substituents of course also interact noncovalently with the active sites of the target enzymes during acylation, thereby having specific effects on the acylation rates. Superimposed crystal structures of a noncovalent complex of a penicillin and that of an analogous D-Ala-D-Ala peptide, both with a LMMB DD-peptidase (the Streptomyces R61 DDpeptidase), show the close similarities between the active sitebound ligand structures. (Figure 2).28 The position of the nucleophilic Ser62 is also shown. 2. Slow Deacylation. It is not immediately obvious, chemically, why the acyl−enzyme intermediate derived from a β-lactam, 1, should be intrinsically less reactive with water than that derived from a peptide, 2 (Scheme 5). It was suggested, however, many years ago that slower deacylation of 1 could be derived from steric hindrance of nucleophilic attack of water in the active site by the pendent heterocycle, which has opposite stereochemistry at the α-position to the normal D-methyl group.29 It was recognized, however, that this would require participation by the enzyme in some fashion,30 to enforce the steric barrier, for example. In the absence of crystal structures, however, no specific progress in this matter was possible. The first atomic resolution crystal structure of a DDpeptidase/β-lactam covalent complex, that of the Streptomyces R61 DD-peptidase (LMMB) with cephalothin, was obtained by Kelly, Knox, and co-workers and published in 1995.31 A

Scheme 4

re-form the free enzyme. In terms of Scheme 4, therefore, an effective β-lactam inhibitor will rapidly acylate the enzyme yielding an acyl−enzyme that only slowly hydrolyzes. These two requirements are addressed separately below for DDpeptidases. 1. Rapid Acylation. This aspect has been extensively discussed for many years. First, relatively high chemical reactivity of the β-lactam is required,23−26 although not so high that there is serious competition with spontaneous hydrolysis in solution.24 The requirement of reactivity is then complemented by the resemblance between the β-lactam and the C-terminal D-Ala-D-Ala moiety of a specific peptide substrate to ensure recognition by the active site. The distinct structural resemblance between a penicillin and the C-terminal D-Ala-D-Ala of a stem peptide (Figure 1, left, ground state structures) was first recognized by Tipper and Strominger.11 As seen in the figure, although the structures are quite similar, a significant difference between the peptide and penicillin lies in the geometry of the reactive amide nitrogen (indicated by an arrowhead), planar in the peptide and quasi-tetrahedral in the B



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above the active site and is important in catalysis (see below). The dihydrothiazine carboxylate is hydrogen bonded to the hydroxyl groups of Thr299 and Thr301. As was pointed out in the paper referred to above,31 the structure clearly shows steric hindrance of approach of a nucleophile at the appropriate angle of attack32 by C2H and N3 of the dihydrothiazine ring (Figures 3 and 4A). Thus, no appropriately placed hydrolytic water

Figure 2. A stereodiagram of superimposed noncovalent (Michaelis) complexes of a penicillin (PDB code 1PW1, aqua) and an N-acyl-Dalanyl- D-alanine (PDB code 1IKG, atomic colors) with the Streptomyces R61 DD-peptidase. α-C atoms of the active site residues shown in Figure 3 were superimposed. The side chain in each case is N-glycyl-L-α-amino-ε-pimelyl.

Scheme 5 Figure 4. Steric hindrance by the pendent heterocycle (van der Waals radii of N3 and C2 are shown) of nucleophilic approach to the carbonyl group of acyl−enzymes derived from reaction of the R61 DDpeptidase with (A) cephalothin (PDB code 1CEG) and (B) benzylpenicillin (PDB code 1PWC).

molecule was observed in the crystal structure. A structure of the same enzyme with benzylpenicillin covalently bound leads to a similar conclusion (Figure 4B).28 This point was also noted with respect to complexes between β-lactams and other DDpeptidases, e.g., E. coli PBP5.33 Some 50 such structures with different β-lactams and different DD-peptidases have now been added to the RCSB Protein Data Bank, and all show essentially the same features, where the pendent heterocycle prevents facile hydrolysis of the acyl−enzyme. Two such examples, featuring HMM DDpeptidases, are shown in Figure 5. This steric hindrance has been quantified by means of the sum Σ of the dihedral angles abcd and bcde shown in 3.18,28 The average value for this

diagram of the active site, taken from the crystal structure, showing the bound cephalothin is shown in Figure 3. This diagram shows the conserved elements of the DD-peptidase active site, viz. the nucleophilic serine, as part of a S1XXK1 motif and the S2XN and K2(H)T(S)GZ motifs (Scheme 2) where Z in the latter motif is usually restricted (see below). This LMMB enzyme is also characterized, uniquely, by Arg285, which hovers

Figure 5. Acyl-enzyme structures of covalent complexes of β-lactams with HMM DD-peptidases: carbenicillin with Pseudomonas aeruginosa PBP3 (PDB code 3OCL) and cefuroxime with Streptococcus pneumoniae PBP2x (PDB code 1QMF).

Figure 3. Structure of the acyl−enzyme derived from reaction of the R61 DD-peptidase with cephalothin (PDB code 1CEG). C



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parameter from the structures available is 75 ± 17°. For the R61 DD-peptidase structures cited above the values of Σ are 56° and 84° for cephalothin and carbenicillin, respectively. At some value of Σ, below 40° perhaps, the acyl−enzyme will become susceptible to hydrolysis (see discussion of βlactamases below) and the β-lactam would no longer be a DD-peptidase inhibitor or an antibiotic.

the N-sulfonate and these same residues also leads to steric hindrance of hydrolysis, e.g., in the case of Pseudomonas aeruginosa PBP3 where Σ = 109°. The fourth residue of KT(S)GZ in HMMA enzymes is more variable and may lead to a more mobile heterocycle. Crystal structures of E. coli PBP5 in covalent complexes with a peptidoglycan-mimetic penicillin and a cephalosporin show the open β-lactam bound to the active site serine and the acylserine carbonyl group bound in the oxyanion hole.35 In the case of the penicillin, the thiazolidine carboxylate is in hydrogen bond contact distance with Ser214. With the cephalosporin, the corresponding carboxylate appears hydrogen bonded to both Ser214 and Arg248, the latter of which has moved across to make contact. In both cases, no clear electron density was observed for the side chain, giving the impression of a less than tightly held ligand. It is interesting and probably relevant to note that the LMMA enzymes are observed to catalyze the hydrolysis of the acyl−enzyme derived from benzylpenicillin 2−3 orders of magnitude more rapidly than those of the other classes described above; well-studied examples are PBP5 of E. coli36 and PBP4 of S. aureus.37 It is also important to the value of Σ and thus to the stability of the β-lactam-derived acyl−enzyme that the angle abcd is found in crystal structures to be 108 ± 13° rather than ∼55° in the free model compound. The value of this angle in the enzyme complexes appears to derive from the optimal requirement of four hydrogen bonds between the ligand and the enzyme. These are between the carbonyl group of the acylserine and the oxyanion hole, particularly to the backbone NH of Z, and between the β-lactam side chain amide group and the backbone carbonyl of Z and the side chain NH of the conserved S2XN asparagine (see, for example, Asn161 in Figure 3). It seems likely that all of the hydrogen bonding interactions noted above that lead to inhibition of DD-peptidases by βlactams must also be required for normal peptide turnover by these enzymes (see below). Recently, the steric hindrance model of DD-peptidase inhibition by β-lactams has been directly supported by results from the boronic acid 4 (Figure 6).38 If the model were correct, one would predict that just as the acyl−enzyme 5 cannot easily

An important question that can be addressed at this stage is why Σ is restricted to values of >40° in the inhibitory complexes. There are two points here, one intrinsic to the βlactam, the other to the enzyme. First, for steric reasons the angle bcde must open up after the β-lactam ring is cleaved. In the intact β-lactam, the bcde angle would be close to 0°. After ring opening, steric interaction between atoms b and e and their substituents forces the bcde dihedral angle to open up. A computational model of a penicilloate in vacuo, after molecular mechanics energy minimization and a molecular dynamics simulation, suggested a value of the bcde dihedral of −70 ± 10° at 300 K. This agrees well with the value of −74° from a crystal structure.34 In penicillin complexes of DD-peptidases, the average of this dihedral was −29 ± 12° (11 structures), and for all cases, including cephalosporins and carbapenems, the average was −33 ± 15°. In all cases, therefore, the enzyme has a significant effect on the bcde angle after ring-opening. Inspection of crystal structures reveals the most important reason for this. In essentially all HMM, LMMB, and LMMC DD-peptidases, the amino acid residue following the conserved KT(S)G motif is threonine or serine and in crystal structures of covalent β-lactam adducts, the CO2− of the pendent heterocycle (see 1 in Scheme 5 and Figures 3 and 5) is hydrogenbonded between the hydroxyl groups of the two Thr/Ser residues. These interactions firmly position the heterocycle with the observed bcde angles. With acyl−enzymes derived from monocyclic β-lactams such as aztreonam, interaction between

Figure 6. Modeled structure showing the steric clash between N3 (blue) and C2 (green) of the thiazolidine and the α-OH (red) of a tetrahedral intermediate derived from attack of water on the acyl−enzyme derived from reaction between E. coli PBP4 and benzylpenicillin. The analogous reaction of the boronic acid 4 is indicated on the left. The model was constructed from the crystal structure of the acyl−enzyme complex between PBP4 and benzylpencillin (PDB code 2EX8). D



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that the R61 DD-peptidase, uniquely, has histidine rather than lysine in the KT(S)G sequence, but it is not clear how this alone would so dramatically affect substrate specificity; there are also R61 analogues that are DD-peptidases but do contain the canonical lysine, e.g., the AmpH DD-peptidase.42) The substrate specificity differences of Scheme 6, therefore, must be expressed in protein structure outside the strictly conserved catalytic core of the active site. Although it is not easy to obtain crystal structures of the acyl−enzyme intermediates of β-lactamase catalysis, a few have been published. For example, Shoichet and co-workers have obtained structures of acyl−enzymes formed on reaction between the AmpC β-lactamase, another class C β-lactamase, and amoxicillin43 and also cephalothin.44 They have also reported the structure of a covalent complex between the cephalosporin loracarbef and a deacylation-defective Q120L/ Y150E mutant of AmpC.45 Figure 8A and Figure 8B show the

reach the tetrahedral intermediate 6 because of steric blocking of the hydrolytic water, then the boronic acid analog 4 could likewise not achieve the boronate adduct structure 7 and thus could not be an inhibitor. As predicted by this rationale, 4 was not an inhibitor of DD-peptidases, although simpler boronic acids lacking the heterocyclic substituent are known to be effective inhibitors through formation of tetrahedral boronate structures by addition of the active site serine hydroxyl group to boron.39,40 The steric hindrance model therefore appears to be firmly established.

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β-LACTAMASES As described above, it is believed that β-lactamases evolved from ancestral DD-peptidases (Scheme 1). The current versions of these two classes of enzyme differ functionally from one another as shown in Scheme 6; DD-peptidases Scheme 6

efficiently turn over D-ala-D-ala terminating peptides (but see ref 41) but not β-lactams and vice versa for β-lactamases. It seems likely from sequence analysis15 that class C β-lactamases (distinguished by a SXXK, YXN, KT(S)G active site) derive from an ancestor of LMMB DD-peptidases while class A βlactamases (SXXK, SXN, KT(S)G) more closely relate to LMMC DD-peptidases. β-Lactamases and thus resistance to βlactams continue to evolve rapidly under the pressure of the βlactams used in human and veterinary clinical practice. Figure 7 shows the superimposed active site residues of a class C β-lactamase (the Enterobacter cloacae P99 enzyme) and a LMMB DD-peptidase (the Streptomyces R61 enzyme). The two structures are clearly very similar from this perspective and it is not possible to decide which is the β-lactamase and which is the DD-peptidase on visual inspection of Figure 7. (It is true

Figure 8. Structures of acyl−enzymes derived from reactions of the AmpC β-lactamase with (A) amoxicillin (PDB code 1LL9) and (B) cephalothin (PDB code 1KVM). The lower structures show the position of (A) the thiazolidine from amoxicillin and (B) the dihydrothiazine from cephalothin, both with the van der Waals radii of N3 and C2 also shown.

amoxicillin and cephalothin structures, respectively, where the lower image in each case shows the approach to the acyl− enzyme carbonyl. It is clear from the latter images that little or no steric hindrance toward nucleophilic attack on the carbonyl would be expected; i.e., amoxicillin and cephalothin would be expected to be substrates of class C β-lactamases, as observed. Quantitatively, the values of Σ from the amoxicillin and cephalothin structures are −15.4° and −49.7°, respectively. Both the abcd (56.9° and 29.8°, respectively) and bcde (−72.3° and −79.5°, respectively) angles contribute to the low value of Σ. A similar situation occurs in the loracarbef structure with abcd and bcde angles of 64.8° and −58.4°, respectively, and thus Σ is 6.4°. The values of the dihedral angles in all three of these cases are closer to those of a free penicilloate than to those bound to a DD-peptidase (see above). In terms of the structures of the complexes, it can be seen for both the penicillin (Figure 8A) and the cephalosporin (Figure 8B) that the pendent heterocycle has swiveled away from Thr316 and now interacts, either directly or via a water molecule, with Asn343 and Arg349. The reason why this is possible with the AmpC β-lactamase but not with the R61 DDpeptidase is evident on inspection of the tertiary structure of

Figure 7. Superimposed active site structures of the R61 DD-peptidase (PDB code 3TPE) and the Enterobacter cloacae P99 β-lactamase (PDB code 1XX2). α-C atoms of the residues shown were superimposed. E



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(Figure 11) that the β-lactam carboxylate can interact with Ser318 and Arg349, the former of these rather than Ser343,

these proteins in Figure 9. The H11 helix of AmpC (Figure 9B) has moved in the direction of the active site and away from the

Figure 9. Secondary structural elements of (A) the R61 DD-peptidase and (B) the AmpC β-lactamase from the crystal structures (PDB codes 3TPE and 2BLS, respectively). Also shown are the side chains of residues discussed in the text.

H1 helix (for labeling of the secondary structural elements of the AmpC and R61 enzymes, see refs 46 and 47, respectively). This brings Arg349 and Asn343 closer to the active site (Figures 9B and 8). The corresponding residues in the R61 DD-peptidase, Val 332 and Ser 326, respectively, remain far from the active site. It is worth noting that the C and N termini of these enzymes are found at the ends of the H11 and H1 helices, respectively.46,47 In the evolution of a β-lactamase from a R61-like LMMB DD-peptidase ancestor, the movement of H11 relative to H1 may therefore not have been difficult. The crystal structures of other DD-peptidases, e.g., the LMMC Actinomadura R39 enzyme and the HMMA S. pneumoniae PBP1b (Figure 10), also do not reveal the alternative carboxylate binding site found in the AmpC β-lactamase (Asn343 and Arg349).

Figure 11. Modeled structure of the acyl−enzyme derived from reaction of the P99 β-lactamase with benzylpenicillin. The model was constructed from the structure of a phosphonate complex (PDB code 1BLS) with energy minimization.

whose side chain is too short. The acyl−enzyme carbonyl is still open to attack (angles abcd = 39.2°, bcde = −68.6°, Σ = −29.6°), and thus the P99 enzyme is a β-lactamase. Note that no crystal structure of an acyl−enzyme derived from this group of enzymes is available. Third generation cephalosporins were developed to resist hydrolysis by class C β-lactamases and are known to form quite stable acyl−enzymes by interaction with these enzymes,48 e.g., kcat = 7 × 10−3 s−1 for the complex of ceftazidine with the P99 enzyme.49 A crystal structure of the ceftazidime complex with AmpC β-lactamase45 (Figure 12) shows how the bulky side chains of third generation cephalosporins restrict the mobility

Figure 10. Secondary structural elements of the Actinomadura R39 DD-peptidase (PDB code 1W8Q) and Streptococcus pneumoniae PBP1b (PDB code 2BG1). The side chain of the nucleophilic active site serine is shown in each.

Class C β-lactamases appear to occupy two main groups. The first, exemplified by the AmpC enzyme of E. coli, is distinguished by the combination R349, N343, and KT(S)GZ where Z is alanine, described above. In this case, as also discussed above, the carboxylate group of the β-lactam-derived acyl−enzyme is able to swing away from the reaction center and interact with R349 and N343. In the other group of class C β-lactamases, comprising some E. coli enzymes and many from Enterobacter and Citrobacter species, the corresponding amino acids are R349, S343, and KT(G)GS. In this case, modeling of the Enterobacter cloacae P99 β-lactamase, for example, shows

Figure 12. Structure of the acyl−enzyme formed on reaction of the AmpC β-lactamase with ceftazidime (PDB code 1IEL). Steric hindrance to attack at the carbonyl is shown on the lower right. F



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peptidase that has been catalytically incapacitated by a methylene bridge between Lys 65 and Tyr159.56 It was assumed that this modification did not significantly affect noncovalent association of the substrate with the active site. The position of the substrate at the active site is shown in Figure 14. The side chain of the substrate containing the glycyl-

of the dihydrothiazine ring in the acyl−enzyme leading to blockage of nucleophilic attack by water45 and thus the very slow turnover as noted above; abcd = 101.2°, bcde = −49.4°, Σ = 51.8°. In this structure, the dihydrothiazine carboxylate is hydrogen bonded to the side chain of Asn 346. Most other βlactam-based inhibitors of β-lactamases, including carbapenems,50 undergo more substantial covalent or noncovalent rearrangement such that the inhibition does not derive solely, if at all, from steric hindrance of deacylation.51,52 Avibactam, a new class of β-lactamase inhibitor, appears to inhibit class C βlactamases by rearrangement into a chemically inert carbamate, although hydrolysis of this also seems to be sterically impeded.53

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WHY CAN’T DD-PEPTIDASES ALSO BE EFFECTIVE β-LACTAMASES? As noted above, on the basis of active site structures (Figure 7), one might anticipate that DD-peptidases would have significant intrinsic β-lactamase activity. Another example of this point is seen in Figure 13 where a superimposition of the active site

Figure 14. Active site structure of the R61 DD-peptidase with a specific peptide substrate noncovalently bound (PDB code 1IKG).

L-α-aminopimelyl

moiety is not shown for simplicity, although it is strongly bound into a specificity site.56 The carboxylate of the terminal D-alanine of the substrate is hydrogen-bonded to Arg285 and Thr299, the carbonyl of the scissile bond is placed in the oxyanion hole, and the “side chain” amide group is hydrogen-bonded to the usual residues found in β-lactam acyl− enzyme structures in both DD-peptidases and β-lactamases, as described above.28,31,44,45,54 Although the C-terminal carboxylate of the substrate appears strongly bound to the protein, it must nevertheless be able to move as the chemical reaction occurs. This requirement is shown in Figure 15A and Figure 15B. Figure 15A shows a modeled structure derived from the crystal structure of a phosphonate transition state analogue covalently bound at the active site of an un-cross-linked enzyme,57 representing the first tetrahedral intermediate formed on attack of the active site serine hydroxyl on a peptide substrate. Accordingly, the nitrogen has a tetrahedral geometry and, according to stereoelectronic principles,58,59 has the nitrogen lone pair antiperiplanar to the just formed C−O bond (Scheme 7). In order for the leaving group nitrogen to be efficiently protonated by Lys65/Tyr159, the tetrahedral intermediate must either invert at nitrogen or rotate about the C−N bond. Both of these, particularly the latter, will be impeded by strong interactions between the terminal carboxylate and the enzyme. A nitrogeninverted structure, ready for protonation (Scheme 7) is shown in Figure 15B. Noticeable is the movement of the carboxylate in Figure 14 → Figure 15A → Figure 15B, but in each case it remains well hydrogen-bonded to the flexible Arg285, to Thr299 and, in 14B, also to Thr301; facile inversion requires stabilization of the carboxylate along the reaction coordinate and, particularly, in the inversion transition state where the nitrogen is effectively planar.

Figure 13. Superimposed active site structure of the P99 β-lactamase (PDB code 1XX2) and S. pneumoniae PBP2x (PDB code 1QME). α-C atoms of the residues shown (except for S2/Y) were superimposed.

elements of the P99 β-lactamase and a HMMB DD-peptidase (Streptococcus pneumoniae PBP2x54) is presented. PBP2x, however, like all HMM DD-peptidases has essentially no βlactamase activity, e.g., kcat values of ∼10−5 s−1.54 As also noted above, the specificity differences of Scheme 6 must arrive from enzyme structural differences outside the catalytic reaction centers shown in Figures 7 and 13. These differences between the R61 DD-peptidase and class C β-lactamases are discussed above. It must be concluded, therefore, that the structural characteristics of an enzyme leading to β-lactamase activity cannot be incorporated into the DD-peptidase structure without impairing the essential DD-peptidase function. This point is discussed below, again in terms of available crystal structures. It has not been easy to obtain a good approximation to the crystal structure of a DD-peptidase bound to a good substrate. Probably the best, one of few, is that of a good substrate, glycyl55 L-α-amino-ε-pimelyl-D-alanyl-D-alanine, bound to a R61 DDG



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changes also permitted diversion of the carboxylate of a tetrahedral intermediate in peptide hydrolysis (Figure 15C), protonation of the amino acid nitrogen of the leaving group would be less efficient and DD-peptidase turnover would suffer. It seems likely, therefore, that this requirement of a mobile carboxylate for the hydrolysis of an acyl−enzyme derived from a β-lactam also leads to a more loosely held tetrahedral intermediate in DD-peptidase catalysis and thus incompatibility of the two reactions in these enzymes. The nature of Z in the active site KT(S)GZ motif is of considerable importance in light of the above discussion. Although it is clearly of use in β-lactamases for diversion of the pendent heterocycle in acyl−enzymes derived from β-lactams (Figure 8), the reason for its presence in virtually all HMM DD-peptidases is not at all obvious. In the currently available structures of these enzymes, it appears only to aid the inhibition by β-lactams (Figures 3 and 5). If this were its only role, it would be rapidly removed by evolution. Since it has not been eliminated, one must conclude that it plays an important part in the essential reaction of the HMM DD-peptidases, the transpeptidation reaction. Direct interaction with a transpeptidase substrate would be the most obvious possibility. Unfortunately, there are at present no crystal structures showing the position of the incoming amine nucleophile in the transpeptidase reaction (Scheme 3). The same is true of LMMC DD-peptidases which are believed to be DDendopeptidases20,41,65 and thus also react with substrates with extended amine leaving groups, lysine and D-aminopimelyl derivatives, in particular. In LMMB DD-peptidases, the enzyme of Streptomyces R61, and its orthologs, Z again is Thr. Although the in vivo role of these enzymes is not known, it has been well established in vitro that the R61 enzyme catalyzes not only DDcarboxypeptidase reactions but also transpeptidase reactions with short peptidoglycan-mimetic amines as acyl acceptors, glycyl dipeptides, for example.66,67 Again, no crystal structures with extended peptide substrates are available, but modeling gives no indication that Thr 301 (Z) is required for turnover of gly-L-amino acid peptides. More extended peptidoglycanmimetic acyl acceptors were not reactive,68 however, suggesting that a true transpeptidase or endopeptidase reaction may not be catalyzed by these enzymes in vivo. They may be pure carboxypeptidases or, perhaps, β-lactam traps,69 where Thr301 would indeed be useful. The LMMA DD-peptidases differ from the classes above in that Z = Thr(Ser) is not essential and, in fact, the amino acid at this position can be quite variable. For example, E. coli PBP5 and its close orthologues have Z = His and an N. gonorrhoeae PBP4 group contains Z = Tyr(Phe). On the other hand, other LMMA enzymes, e.g., E. coli PBP6, S. pneumoniae PBP3, and S. aureus PBP4 do retain Z = Thr(Ser). Apparently, Z = Thr(Ser) is not required for the in vivo role of LMMA DD-peptidases, and this is in accord with evidence that these enzymes are purely DD-carboxypeptidases.70,71 If it were given that Z = Thr/Ser is required for transpeptidase/endopeptidase activity, Figure 16 shows how it may be required for direct interaction with an extended nucleophile (transpeptidase reaction) or leaving group (endopeptidase reaction). It shows the tetrahedral intermediate arising from attack of a D-aminopimelyl N-terminus on the carbonyl of a specific peptide substrate of the R39 DDpeptidase carbonyl, where the carbonyl of the penultimate Dalanyl residue of the nucleophilic stem peptide can be

Figure 15. Modeled stereochemistry of tetrahedral intermediates (TI) formed in the reaction between the R61 DD-peptidase and a specific peptide substrate. These models were constructed from the structure of a complex of the enzyme with an analogous phosphonate inhibitor (PDB code 1MPL). Lone pairs on the leaving group nitrogens are shown in aqua coloring.

Scheme 7

The issue of nitrogen inversion in the tetrahedral intermediates of amidohydrolase catalysis has been discussed for some time along the lines described above.58,60−63 In particular, the importance of stabilization of the inversion transition state by alternative hydrogen bonding has been noted.63 Conversely, a nice model study by Rebek and coworkers64 shows how a single immobile hydrogen bond can significantly increase the inversion barrier of an amine trapped in a confined space. Now, as discussed above, class C β-lactamases have undergone structural changes to allow additional functional groups to divert the β-lactam carboxylate after acylation and thus allow acyl−enzyme hydrolysis. If these same structural H



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lactams but that also hydrolyzed peptidoglycan stem peptides through residual DD-peptidase activity. The second point has also been discussed in depth elsewhere. High DD-peptidase activity against small peptidoglycan-mimetic peptides is only seen in LMMB and LMMC DD-peptidases that have specific binding site for elements of peptidoglycan structure.41 It has been suggested that the LMMC enzymes, at least, may be sensors for molecules containing these elements.77,78 Clearly the interactions with this site strongly promote the DD-peptidase reaction.70 Other DDpeptidases, including the biosynthetically important HMM and LMMA enzymes, have little or no activity against small peptidoglycan-mimetic peptides.41 The structural basis for their in vivo activity is still not well understood. As far as is known, these specificity elements are also not present in β-lactamases except perhaps in vestigial form.79,80 The factors described above, taken together, are sufficient to explain why the serine β-lactamases of today have little DDpeptidase activity.

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CLASS A β-LACTAMASES As noted above, class A β-lactamases probably derive from LMMC DD-peptidases because they have the SXN rather than the YXN active site motif (Scheme 2 and Figure 16).81 These enzymes are also able to effectively catalyze the hydrolysis of acyl−enzymes derived from β-lactams (Scheme 6). Figure 17

Figure 16. Modeled structure of the tetrahedral intermediate formed from reaction of a specific acyl−enzyme with the amine group of a Daminopimelyl acyl acceptor. The acyl−enzyme derives from reaction of the Actinomadura R39 D-peptidase with a specific peptide substrate. The tetrahedral intermediate model structure was obtained from the structure of this enzyme with an analogous specific boronate inhibitor (PDB code 2XDM) by means of MD simulations and energy minimization. For clarity, the carbon atoms of the extended acyl acceptor are shown in orange.

hydrogen-bonded to the hydroxyl group of Thr413. This LMMC enzyme is able to catalyze transpeptidase and endopeptidase reactions as well as carboxypeptidase.65 The noted hydrogen bond of Figure 16 is also possible with a Llysine N-terminus as nucleophile. Such a required hydrogen bond may explain the essential Z = Thr/Ser of HMM DDpeptidases. It is known that present day well-evolved serine β-lactamases are very poor DD-peptidases (Scheme 6).72 One likely reason for this is the shape of the active site beyond the reaction center shown in Figures 7 and 13. The preference of β-lactamases vs DD-peptidases for substrates bearing quasi-tetrahedral nitrogen atoms in the scissile bond has been demonstrated with Nacylaziridines.73 There are other factors, however, which are noted below. First, as discussed in detail elsewhere,18,74,75 β-lactamase evolution from DD-peptidases also included as an important factor selection against the D-methyl group of the penultimate D-alanine of the peptidoglycan stem peptide. This methyl group stands out as a distinct structural difference between D-alanyl-Dalanine terminating peptides and classical β-lactams (Figures 1 and 2). DD-peptidases have a binding pocket available for this methyl group, which is closed off in β-lactamases. Thus, Dalanyl-D-alanine peptides noncovalently interact weakly with βlactamases and modeling shows tetrahedral intermediates of acylation by D-alanyl peptides are poorly stabilized by these enzymes (ref 74, ref 76, and Pratt, R. F., unpublished). This feature of β-lactamase evolution was probably an important early step, since it would be nonproductive for a bacterium to produce large quantities of an enzyme that did destroy β-

Figure 17. Modeled structure of the acyl−enzyme formed on reaction of the class A Toho β-lactamase with cephalothin. This model was constructed from the structure of a noncovalent complex of cephalothin with the E166A mutant of the enzyme (PDP code 1IYP) with energy minimization; Glu166 has been reinserted.

shows a model of an acyl−enzyme formed between cephalothin and a class A β-lactamase (TOHO-1). This model is derived from the crystal structure of a complex between cephalothin and an E166A mutant of the enzyme, which is deacylation defective (see below).82 In this structure, the substrate is covalently attached to Ser70 and interacts with the usual active site components, all as expected. Of interest here is the position of the dihydrothiazine carboxylate, where the oxygen atoms are within hydrogen bonding distance of both Thr235 OH and Ser237 OH, much as is found in the HMM DD-peptidases. The dihedral angles (abcd = 100°, bcde = −39.6°, Σ = 60.4°) are such as to suggest severe steric hindrance of hydrolysis, again as for DDpeptidases (Figures 3 and 4). Evolution of the class A βI



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peptidase that, along with β-lactamases, form a protein superfamily essentially restricted to bacteria. There are, however, a few functional variants derived from the LMMB DD-peptidases that appear to be D-amino acid amidases and peptidases.86−88 Crystal structures of these bacterial enzymes clearly show the fold and most of the active site residues of a LMMB DD-peptidase.87,89,90 Somewhat more distant from the LMMB DD-peptidases than the enzymes described above are another group of bacterial enzymes, the family VIII carboxylesterases. These include EstA,91 EstB,92 EstC,93 EstU1,94 and EstY29,95 all of which efficiently hydrolyze hydrophobic esters, for example, those of medium sized fatty acids. They have little or no βlactamase activity except for EstC, which has modest turnover activity against nitrocefin although none against classical βlactams.93 Amino acid sequence analysis shows that the classical β-lactamase active site motifs are significantly modified; SXXK is uniformly present, YXN is present as YXX, and K(H)T(S)GT(S) is present as WGGZ or WSGZ (EstY29), where Z is nonpolar. The absence of K(H)T(S) suggests that these enzyme are neither β-lactamases nor DD-peptidases, since these residues interact with the substrate terminal carboxylate, as discussed above. Crystal structures of EstA,96 EstB,92 Est U1,97 and EstY2998 show the general protein fold of a LMMB DD-peptidase/class C β-lactamase, although there are differences. The active sites are narrower and less polar, appropriate for their esterase substrate. EstU1, however, is apparently also inhibited by β-lactams with which it can form stable acyl− enzymes. The crystal structure of a covalent complex of this enzyme with cephalothin has been published.97 It shows the βlactam, ring-opened, covalently bound to the active site serine very much as in a DD-peptidase (Figure 3). The acyl-serine carbonyl is in the usual oxyanion hole, and the serine oxygen is attended by lysine and tyrosine residues as by Lys65 and Tyr159 in the R61 DD-peptidase.31 The dihydrothiazine ring is held in a position to sterically hinder hydrolysis (abcd = 100.8°, bcde = −52.6°, Σ = 48.2°) just as in the DD-peptidase. It is held there by strong interactions between the heterocyclic carboxylate and Arg408. This residue is brought close by the positioning of a helix, much as in a class C β-lactamase (Figure 9). In this case, however, an arginine has been placed that hinders movement of the heterocycle carboxylate rather than facilitating it. Arginine 408 is not present in EstA or EstB;

lactamases proceeded, however, not in a way leading to the swiveling away of the dihydrothiazine to allow attack by water but by incorporation of a new functional group, Glu166, into a remodeled Ω loop.81 This residue, aided usually by Asn170 to hold a specific water molecule and acting as a general base, is able to catalyze rapid hydrolysis of the acyl−enzyme in a way not available to class C β-lactamases.83,84 Thus, in class A βlactamases, the attack of water on the acyl−enzyme is believed to occur on the Si face (from the protein interior), whereas in class C β-lactamases, attack occurs on the Re face (external) (Scheme 8). Scheme 8

Although the incorporation of Glu166 provides a very effective method of catalyzing deacylation of acyl−enzymes derived from β-lactams, it is not a viable strategy for D-alanyl peptides, since the D-methyl group would hinder both acylation (Figure 18) and water attack on the Si face during deacylation. Indeed, class A β-lactamases are very poor catalysts of the hydrolysis of acyl-D-ala-D-ala peptides.72 It would not be a useful catalyst of the transpeptidation reaction either, since the large (peptidoglycan) nucleophile in that case must approach the external (Re) face. Thus, the strategy employed by class A β-lactamases to catalyze acyl−enzyme hydrolysis would not be effective in DD-peptidase catalysis. The same conclusion can be drawn for class D β-lactamases, which are also poor catalysts of D-ala peptide hydrolysis.72 In this case, a carbamate of the SXXK lysine is thought to act as a catalyst of both acylation and deacylation, the latter by water attack from the Si face as for class A β-lactamases.85

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DD-PEPTIDASE EVOLUTION: OTHER PATHS Apart from the β-lactamases, there appear to be no other known descendants of the HMM, LMMA, and LMMC DD-

Figure 18. Stereodiagram of a modeled structure of a covalent N-acylglycyl phosphonate transition state analogue (PDB code 1AXB) at the active site of the class A TEM-1 β-lactamase, which has been converted to an N-acyl-D-alanyl structure by manual addition of the D-methyl group. J



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evolve in terms of the mechanisms of β-lactam inhibition of DD-peptidases and β-lactam hydrolysis by β-lactamases, revealed in each case by the crystal structures of these enzymes obtained over the past 20 years. The major points discussed are the following: 1. β-Lactams inhibit DD-peptidases by the formation of inert acyl−enzymes and are thus antibiotics. These complexes are inert to hydrolysis because of steric hindrance to deacylation by the pendent heterocycle. The heterocycle carboxylate is held in place by the same functional groups needed to bind to the D -Ala carboxylate of the leaving group in the ground and transition states of the transpeptidation reaction of peptidoglycan synthesis. 2. Class C β-lactamases evolved from a DD-peptidase ancestor by bringing in new functional groups to divert the heterocycle carboxylate in the acyl−enzyme complex from hindering deacylation. 3. Class A β-lactamases evolved by bringing in a new deacylation catalyst (Glu166) that promotes deacylation of β-lactams by nucleophilic attack from the protein interior side of the acyl carbonyl. 4. Neither method of facilitated β-lactam deacylation is compatible with an efficient transpeptidation reaction. 5. Thus, DD-peptidases cannot efficiently catalyze deacylation of β-lactams while retaining their transpeptidation ability and a new and different class of enzyme (βlactamases) that efficiently catalyzed β-lactam hydrolysis necessarily had to evolve. Thus, β-lactamases: why and how.

whether it has a role with a natural substrate of EstU1 is not known. Only very limited expansion of the LMMB DD-peptidase fold into metazoa seems to have occurred. For example, esterase-like LACTB proteins were discovered from sequence data in nematodes and echinoderms and also in vertebrates, including humans.99 The LACTB proteins retain the SXXK, YXX, and HTG motifs of the R61 DD-peptidase. Their role in biology is not yet well understood, but it almost certainly does not involve β-lactams.100,101

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SERINE β-LACTAMASES FROM DD-PEPTIDASES: RECENT EVOLUTION? As mentioned above, serine β-lactamases are thought to have evolved from DD-peptidases some three billion years ago (Scheme 1).102 There is no evidence of “second thoughts”, where β-lactamase activity has been selected for from more recent DD-peptidases.15,103 Such, more recent, evolution of DD-peptidases as seems to have occurred centers on producing enzymes that have lower affinity for β-lactams, i.e., are acylated by them more slowly.104 Under the selective pressure of several generations of β-lactams used clinically over the past 70 years, β-lactamase evolution has proceeded apace.105 Interestingly, class A and class D β-lactamases appear to be evolving more rapidly than class C under these conditions. At least one significant and explicit effort seems to have been made to artificially induce transformation of a DD-peptidase into a β-lactamase. Peimbert and Segovia attempted a process of directed evolution of a HMMB DD-peptidase, PBP2x of Streptococcus pneumoniae by mutation of residues close to the active site that appeared to them to represent distinctive features of class A β-lactamases as opposed to DDpeptidases.106 This procedure produced one particular mutant that had significantly higher activity against third generation cephalosporins. No such enhancement of rates of hydrolysis of other β-lactams was, however, observed. It is possible that the selective effect on third generation cephalosporins simply arose from increasing the mobility of the bulky third generation side chains (see Figure 12) rather than from a general process allowing the pendent heterocycle to move into a more hydrolytically productive conformation (Figure 8). In other research, not specifically directed toward evolution of βlactamase, certain active site mutations of E. coli PBP5 were found to exhibit modest enhancement of β-lactam turnover.107 Since this enzyme and its orthologues have the highest known β-lactamase activity of any DD-peptidase (kcat = 3 × 10−3 s−1 107), they seem the most obvious choice as a candidate for directed evolution. The retroevolution of a DD-peptidase from a β-lactamase, chemically the more difficult direction, has also been attempted. Prior to the availability of crystal structural information, Richards and co-workers replaced a 28 amino acid sequence, including, but not replacing, the nucleophilic serine of the active site of the TEM-1 β-lactamase with the corresponding segment from E. coli PBP5.108 Astonishingly, particularly in retrospect, the mutant was reported to achieve 1% of the (admittedly low) DD-peptide hydrolase activity of PBP5 against N,N′-diacetyl-L-lysyl-D-alanyl-D-alanine.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: 860-685-2629. E-mail: [email protected]. Notes

The author declares no competing financial interest. Biography Rex F. Pratt received his B.Sc. (Hon) and Ph.D. degrees from the University of Melbourne, Australia. After postdoctoral work with Thomas Bruice, Gordon Lowe, and Bert Vallee of the University of California, Santa Barbara, Oxford University, U.K., and Harvard Medical School, respectively, he joined the faculty of Wesleyan University where he is J. W. Beach Professor of Chemistry. He is a mechanistic enzymologist whose research program includes studies of the kinetics and mechanism of DD-peptidase and β-lactamase catalysis and of the reactions of these enzymes with novel substrates and inhibitors.

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ACKNOWLEDGMENTS The author’s research referred to in this review was supported by National Institutes of Health Grant AI-17986 and by Wesleyan University.

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ABBREVIATIONS USED HMM, high molecular mass; LMM, low molecular mass

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REFERENCES

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CONCLUSIONS The aim of this review was to compile the elements of our understanding of why it was necessary for the β-lactamases to K



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