The Crystal Structure of a Penicilloyl-serine Transferase of Intermediate Penicillin Sensitivity

The serinedd-transpeptidase/penicillin-binding protein ofStreptomyces K15 catalyzes peptide bond formation in a way that mimics the penicillin-sensitive peptide cross-linking reaction involved in bacterial cell wall peptidoglycan assembly. TheStreptomyces K15 enzyme is peculiar in that it can be considered as an intermediate between classical penicillin-binding proteins, for which benzylpenicillin is a very efficient inactivator, and the resistant penicillin-binding proteins that have a low penicillin affinity. With its moderate penicillin sensitivity, theStreptomyces K15 dd-transpeptidase would be helpful in the understanding of the structure-activity relationship of this penicillin-recognizing protein superfamily. The structure of theStreptomyces K15 enzyme has been determined by x-ray crystallography at 2.0-Å resolution and refined to anR-factor of 18.6%. The fold adopted by this 262-amino acid polypeptide generates a two-domain structure that is close to those of class A β-lactamases. However, the Streptomyces K15 enzyme has two particular structural features. It lacks the amino-terminal α-helix found in the other penicilloyl-serine transferases, and it exhibits, at its surface, an additional four-stranded β-sheet. These two characteristics might serve to anchor the enzyme in the plasma membrane. The overall topology of the catalytic pocket of the Streptomyces K15 enzyme is also comparable to that of the class A β-lactamases, except that the Ω-loop, which bears the essential catalytic Glu166residue in the class A β-lactamases, is entirely modified. This loop adopts a conformation similar to those found in theStreptomyces R61 dd-carboxypeptidase and class C β-lactamases, with no equivalent acidic residue.

The bacterial cell wall peptidoglycan assembly machinery comprises, among other components, multimodular (transglycosylase/transpeptidase) membrane enzymes that catalyze the polymerization of lipid-transported disaccharide-pentapeptide units. Cross-linking of peptidyl moieties of adjacent glycan strands is a two-step reaction. The first step involves the rupture of the D-alanyl-D-alanine bond of a peptide unit precursor acting as carbonyl donor, the release of the carboxyl-terminal D-alanine, and the formation of a serine ester-linked peptidyl enzyme. The second step involves the breakdown of the peptidyl enzyme and the formation of a new peptide bond between the carbonyl of the D-alanyl moiety and the -amino group of another peptide unit acting as amino acceptor. The enzyme is classified as a DD-transpeptidase because the susceptible peptide bond of the carbonyl donor extends between two carbon atoms with the D-configuration.
Penicillins, the most familiar antibacterial agents, exert their effects by inactivating the serine DD-transpeptidase modules of the peptidoglycan-synthesizing enzymes. Because penicillin is a cyclic analogue of the D-alanyl-D-alanine-terminated carbonyl donors, the reaction stops at the level of the serine ester-linked penicilloyl enzyme, and the enzymes behave as penicillin-binding proteins (PBPs). 1 Resistance to ␤-lactam antibiotics is PBP-and ␤-lactamasemediated. PBPs with a reduced affinity for the drug result from local changes in the amino acid sequences of the wild-type forms. Serine ␤-lactamases hydrolyze penicillin into penicilloate via the formation of a hydrolytically labile serine esterlinked penicilloyl enzyme. The emergence of an increasing number of resistant bacteria has become a threatening public health problem. It has fostered interest in understanding the biochemical and mechanistic features of the penicillin targets.
The Streptomyces K15 serine DD-transpeptidase/PBP (hereafter referred to as the K15 enzyme) is a 262-amino acid monomodular protein with a molecular mass of 27,474 Da (1). Although lacking transmembrane segments, the K15 enzyme is associated with the plasma membrane of the wild-type strain. Overexpression of the encoding gene in Streptomyces lividans results in the secretion of ϳ30% of the synthesized enzyme in the culture medium. The cloned enzyme has the same enzymatic properties as the membrane-associated form and requires the presence of 0.5 M NaCl to remain water-soluble (2).
The fate and rate of the two-step transpeptidation reaction catalyzed by the K15 enzyme depend on both the nature of the scissile bond (peptide, ester, thiol ester) of the carbonyl donor and the acceptor activity of an exogenous nucleophile (3). With (R)-D-alanyl-D-alanine-terminated peptide donors and in aqueous media, the released D-alanine is reutilized as an amino acceptor in the enzyme deacylation step so that the peptide donor is continuously regenerated and the enzyme is seemingly silent, although it turns over one time every 10 s. In the presence of amino acceptors structurally related to cell wall peptidoglycan, the peptidyl ((R)-D-alanyl) enzyme intermediate is aminolyzed much more effectively than it is hydrolyzed, and under certain conditions, the acyltransferase functions exclusively as a DD-transpeptidase (4). In contrast, the DD-carboxypeptidases/PBPs have a much increased preference for water as the attacking nucleophile of the peptidyl enzyme.
The K15 enzyme is a PBP. The value of the second-order rate constant of enzyme acylation by benzylpenicillin is ϳ150 M Ϫ1 s Ϫ1 , and the serine ester-linked penicilloyl enzyme undergoes hydrolytic breakdown at an extremely slow rate via two pathways that give rise to benzylpenicilloate and phenylacetylglycine, respectively. Cefoxitin is a better inactivating agent (rate constant of ϳ850 M Ϫ1 s Ϫ1 ), and in this respect, the K15 enzyme differs from many PBPs for which benzylpenicillin is one of the most efficient known inactivators tested (5). Low enzyme acylation rate values confer increased "intrinsic" resistance on the bacterial cell. Thus, for example, the values of the rate constants of enzyme acylation by benzylpenicillin are 10 -20 M Ϫ1 s Ϫ1 for the low affinity PBP2Ј of the methicillin-resistant Staphylococcus aureus and PBP5 of Enterococcus hirae. In contrast, the rate constant values are 18,000 M Ϫ1 s Ϫ1 for the Streptomyces R61 DD-carboxypeptidase/PBP, 58,000 M Ϫ1 s Ϫ1 for Streptococcus pneumoniae PBP2x, and 300,000 M Ϫ1 s Ϫ1 for Bacillus licheniformis PBP1 (6 -9). In comparison with those extreme values, the K15 enzyme may be considered as a PBP of intermediate penicillin sensitivity.
The PBPs and the serine ␤-lactamases are members of the penicilloyl-serine transferase family (10). The three-dimensional structures are known for the class A ␤-lactamases of S. aureus PC1 (11), Streptomyces albus G (12), B. licheniformis 749/C (13), and Escherichia coli TEM (14 -16) and for the two class C ␤-lactamases of Citrobacter freundii (17) and Enterobacter cloacae P99 (18). Among the PBPs, only the structure of the monomodular DD-carboxypeptidase of Streptomyces R61 has been determined at high resolution (19), and that of the multimodular PBP2x from S. pneumoniae has been established at low resolution (20). Understanding the structure-function relationships among members of the penicilloyl-serine transferases requires a detailed knowledge of each type of enzyme of this family at the atomic level. The K15 enzyme is the only serine DD-transpeptidase/PBP that has been characterized in detail in biochemical terms. In this paper, we describe the high resolution x-ray crystallographic structure of this enzyme, compare its structure with that of the class A serine ␤-lactamases, and emphasize the most significant structural features that distinguish these two types of enzymes.

EXPERIMENTAL PROCEDURES
Crystallization and Data Collection-The K15 enzyme is synthesized in the form of a 291-amino acid precursor possessing a cleavable 29amino acid signal peptide. The overproduced soluble enzyme was purified to 95% homogeneity, and the mass spectrum revealed a mass corresponding to the theoretical value of 27,474 Da. Large bipyramidal crystals with maximum dimensions of 0.6 ϫ 0.5 ϫ 0.3 mm were obtained at 20°C within a few days in 10-l drops containing 1.3 mg/ml protein solution in 50 mM Tris-HCl (pH 7.2), 0.4 M NaCl, 10 mM NaN 3 , 0.2 mM dithiothreitol, and 7.5% (w/v) polyethylene glycol 4000 or 8000, equilibrated against a 1-ml reservoir well containing 15% (w/v) polyethylene glycol 4000 or 8000 in the same buffer. The crystals belong to the orthorhombic space group P2 1 2 1 2 1 , with cell dimensions of a ϭ 46.59 Å, b ϭ 54.53 Å, and c ϭ 108.70 Å. They contain 51% solvent and one molecule/asymmetric unit (21).
X-ray diffraction data were collected at 20°C using a Siemens X1000 area detector. The x-ray source was graphite-monochromated Cu ␣ radiation produced by a Rigaku RU-200 rotating anode generator operat-ing at 50 kV and 90 mA with a 0.3-mm fine-focus cathode. Indexing, integration, scaling, and merging of the intensity data were carried out using the XENGEN Version 2.0 (Native 1 and 2 and heavy atom derivatives) (22) and the SAINT (Native 3 and 4) (23) software packages. To improve the completeness of the native data, the four native sets were merged with the BIOMOL package provided by the Groningen-CF/BIOMOL Group (Department of Chemistry and Biophysical Chemistry, Crystallography Group, Rijkuniversiteit Groningen) ( Table I).
Structure Determination and Refinement-The structure was solved by the method of multiple isomorphous replacement with anomalous scattering (MIRAS) using the PHASES program (24). Of the 30 heavy atom derivatives tested, only p-chloromercuribenzoate and mersalyl mercury derivatives showed binding sites. Of the two cysteine residues present in the K15 enzyme at positions 38 and 223, respectively, only one is labeled by p-chloromercuribenzoate in aqueous solution, resulting in a drastic decrease in the peptidase activity and penicillin-binding capacity (25). Table I gives the refinement parameters of the heavy atom derivatives at 3.0-Å resolution. The quality of the 3.0-Å MIRAS electron density map was improved by solvent flattening using the SQUASH program (26).
The protein structure was built stepwise using TURBO-FRODO (27) and refined by simulated annealing using X-PLOR (28). Fourteen cycles of refinement (positions and temperature factors) and model refitting were carried out. At each step, the structure was constructed on the basis of 2F o Ϫ F c and F o Ϫ F c maps computed at a 20-Å lower resolution limit. As long as the model was incomplete, the MIRAS phases were used and combined with those determined by the model in order to improve the quality of the electron density map and to avoid model bias. The structure was refined to 2.0-Å resolution with a conventional Rfactor of 18.6%, and the statistics of refinement are summarized in Table I.
Calculation of the Molecular Hydrophobicity Potential (MHP)-The hydrophobicity of a molecule is usually defined by a single parameter representing its partition coefficient between water and octanol. For the analysis of complex interactions between lipids and proteins, this parameter was extended to the most elaborate concept of helical hydrophobic moment (29). The calculation of the MHP along the peptide sequence was based upon the concept introduced by Furet et al. (30) and Fauchère et al. (31) for a small molecule. This approach was extended to a three-dimensional representation of the molecular envelope with isopotential contour lines around an 18-residue helical peptide (32). We assumed that the hydrophobic interaction between two residues decreases exponentially with the distance between the residues according to the following equation: MHP ϭ ⌺ E tri exp(r i Ϫ d i ), where E tri is the transfer energy of the atom i, r i is the radius of the atom i, and d i is the distance between atom i and a point M.
The transfer energy E tri represents the energy for individual atoms, calculated from the transfer energy compiled by Tanford (33). Assuming that the experimental molecular energy is the sum of the atomic energies, the E tri values are estimated for the different atoms. The MHP for proteins was calculated by a cross-sectional computation method. A plane was moved every 3 Å, and the MHP was computed for all points in the plane defined by a 3-Å side grid.

RESULTS
Overall Structure-The overall three-dimensional structure of the K15 enzyme consists of a single polypeptide chain organized into two domains. One domain contains mainly ␣-helices, and the second one is of ␣/␤-type. The K15 enzyme bears the signature fold topology of the penicilloyl-serine transferase superfamily, but it exhibits more overall similarity to the class A ␤-lactamases (Fig. 1). Using the standard secondary structure numbering of the class A ␤-lactamases (35), the first domain contains a central helix (␣2) that is surrounded by four helices (␣4, ␣5, ␣6, and ␣9) and, in addition, a four-stranded antiparallel ␤-sheet (␤2a to ␤2d) and helix ␣3. The ␣/␤-domain consists of a five-stranded antiparallel ␤-sheet (␤1 to ␤5) that is covered on one face by the short helix ␣10 and the long carboxylterminal helix ␣11 and on the other face by helix ␣8 and one turn of a 3-10 helix (␣0).
Comparison of the K15 Enzyme and the Class A ␤-Lactamases-Optimal superimposition of the K15 enzyme and class A ␤-lactamases was performed using a maximum distance cutoff of 1.2 Å and, as fitted atoms, the equivalent C-␣ atoms of the catalytic residues Ser 35 , Lys 38 , Ser 96 , Lys 213 , and Gly 215 for the K15 enzyme and Ser 70 , Lys 73 , Ser 130 , Lys 234 , and Gly 236 for the class A ␤-lactamases. The structurally superimposable regions comprise 170 amino acids, and the root mean square deviation for the 170 pairs of C-␣ atoms varies from 2.1 to 2.5 Å depending on the ␤-lactamase being compared. As shown in Fig. 2, one may note that pairwise comparison between the amino acid sequences reveals only ϳ15% of strict identity between the K15 enzyme and each of the class A ␤-lactamases.
In comparison with the class A ␤-lactamases, the K15 enzyme exhibits four main structural differences (Fig. 3A): (i) the absence of the NH 2 -terminal helix ␣1; (ii) the insertion of 11 residues after helix ␣5, resulting in the extended helix ␣6; (iii) the absence of helix ␣7, which contains the class A ␤-lactamasespecific EXELN motif; and (iv) the occurrence of a large insert between helices ␣9 and ␣10. This insert contains strands ␤2c and ␤2d of the four-stranded ␤-sheet, and the stability of this ␤-sheet on the surface of the ␣-domain is reinforced by two salt bridges involving Asp 54 -Arg 86 and Asp 81 -Arg 191 .
The MHP analysis (32) of the K15 enzyme and class A ␤-lactamases (Fig. 4) reveals an increase in the hydrophobic isopotential areas in the K15 enzyme, generated by the N-and C-terminal regions that cover the five-stranded ␤-sheet and the loop corresponding to the 20-amino acid insert. This marked The numbers given in parentheses denote the respective resolution limit. b R merge ϭ ⌺⌺͗I͘ Ϫ I i /⌺⌺ I i . c pCMB, p-chloromercuribenzoate; rms, root mean square. d R iso ϭ ⌺ F 2 PH Ϫ F 2 P /⌺ F 2 P ͉, where PH and P are related to the heavy atom derivative and the native enzyme, respectively. e x, y, and z are positions of heavy atoms in fractional atomic coordinates. f B is the isotropic thermal factor, and q is the absolute occupancy. g Phasing power ϭ ͗F H(calc) ͘ /E, where ͗F H(calc) ͘ and E are average structure factor and lack of closure error, respectively. hydrophobic characteristic, which is very different from the homogeneous distribution of the hydrophilic/hydrophobic isopotential areas observed in soluble exocellular proteins, could lead to the interaction of the K15 enzyme with the plasma membrane.
In comparison with the Streptomyces R61 carboxypeptidase/ PBP (Protein Data Bank code 3PTE) and the class C ␤-lactamase of E. cloacae P99 (Protein Data Bank code 1BLS), the K15 enzyme shows a much higher extent of divergence (Fig. 3B). The only common secondary structures are the central helix ␣2 and the five-stranded ␤-sheet. Helices ␣5, ␣6, ␣8, and ␣11 are also present, but they have different lengths and orientations.
Catalytic Pocket-As observed with the other penicilloylserine transferases, the active site of the K15 enzyme is located at the interface between the two domains and is mainly defined by three conserved structural elements (Fig. 5). The center of the catalytic cleft is occupied by the Ser 35 -Thr 36 -Thr 37 -Lys 38 tetrad, which includes the nucleophilic Ser 35 residue at the amino-terminal end of helix ␣2. One side of the cavity is defined by the Ser 96 -Gly 97 -Cys 98 loop connecting helices ␣4 and ␣5. The Lys 213 -Thr 214 -Gly 215 triad lies on strand ␤3 on the opposite side of the cavity. The backbone NH group of the essential Ser 35 residue and that of Ser 216 downstream from the motif Lys 213 -Thr 214 -Gly 215 occupy positions that are compatible with the oxyanion hole function required for catalysis.
With X denoting a variable amino acid, the amino acid sequence signature of the penicilloyl-serine transferases is de-fined by the three active-site structural elements: the invariant S*XXK tetrad, the SX(N/C/S) or YXN triad, and the (K/H/R)(T/ S)G triad. Divergence at the level of the second element might have given rise to the "tyrosine" and "serine" enzyme subgroups, respectively (37). All known PBPs, including the K15 enzyme and the class A ␤-lactamases, belong to the so-called serine subgroup, whereas the Streptomyces R61 DD-carboxypeptidase/PBP and the class C ␤-lactamases belong to the tyrosine subgroup. Superimposition of the active sites reveals that the ␥-OH of the serine residue of the first subgroup and the phenolic OH of the tyrosine residue of the second subgroup occupy equivalent positions with respect to the nucleophilic serine and strand ␤3, respectively (Fig. 6A). As the side chain of a tyrosine residue is bulkier than that of a serine residue, this spatial equivalence must be the result of a reorganization of both the corresponding loops and the interconnecting secondary structures of all the ␣-domains. Accordingly, the serine and tyrosine subgroups show a larger extent of divergence at the level of the ␣-domains than at the level of the ␣/␤-domains.
The top of the catalytic cleft of the K15 enzyme is defined mainly by the Thr 199 -Asn 200 -Gly 201 triad (immediately upstream from helix ␣10), and the bottom of the cleft by the backbone of the Phe 142 -Asp 143 -Gly 144 triad. The Thr 199 -Asn 200 -Gly 201 triad adopts the same topology as in the class A ␤-lactamases (Fig. 6B) despite large structural differences between the helix ␣9/strand ␤3-connecting polypeptide chains.
In the class A ␤-lactamases, the EXXLN motif of the ⍀-loop (which connects helices ␣6 and ␣7) lies at the bottom of the active site, and the side chain of Glu 166 plays an important role in the catalyzed hydrolysis of penicillin. The K15 enzyme has no motif similar to the ⍀-loop, and the fold defined at this position by the Phe 142 -Asp 143 -Gly 144 triad is structurally equivalent to that defined by the Gln 219 -Ala 220 -Tyr 221 triad in the E. cloacae class C ␤-lactamase and by the Ser 236 -Ala 237 -Gly 238 triad in the Streptomyces R61 DD-carboxypeptidase (Fig. 6A).
Solvent Structure-In the crystal structure, the K15 enzyme is solvated by 149 ordered water molecules mainly located in the first coordination shell, directly hydrogen-bonded to protein atoms. In the active site, where a dense hydrogen bond network prevails, the solvent molecules that interact with the essential amino acids (Table II) are the most buried ones and, accordingly, exhibit the lowest temperature factor values. As the charge distribution induced by the side chains varies among the different classes of enzymes, it is obviously difficult to make a rigorous comparison. The unique water contact with the hydroxyl group of the active Ser 35 residue is made with Wat 336 .
This water molecule could be compared with the one found in the oxyanion hole of the class A (Wat 295 for the TEM1 enzyme) and class C (Wat 1087 for the P99 enzyme) ␤-lactamases. However, Wat 336 is not buried so deeply in the catalytic pocket and does not make any interaction with the backbone amide groups of Ser 35 and Thr 214 . In comparison with the other penicilloylserine transferases, the side chain of the nucleophilic serine of the K15 enzyme is displaced through the 1 -dihedral angle rotation. The values of 1 are 52.3°, Ϫ71.5°, Ϫ16.3°, and 56.6°i n the K15, TEM1, R61, and P99 structures, respectively. The shift of the hydroxyl group of Ser 35 toward strand ␤3 and away from the bottom of the active site can certainly be related to the displacement of Wat 336 .
In the same way, the so-called hydrolytic water molecule found in all class A ␤-lactamase structures is not really conserved in the K15 enzyme. This water molecule (Wat 292 in the TEM1 enzyme) forms a bridge between the nucleophilic serine hydroxyl group and the Glu 166 carboxylate side chain. Without the ⍀-loop, the K15, R61, and P99 enzymes cannot exhibit such a water molecule that is tightly bound and a fortiori for the K15 enzyme, which has a different conformation for the active serine. One can only remark that as in the R61 and P99 enzymes, there is in the K15 structure a water molecule (Wat 381 ) that neighbors the class A ␤-lactamase hydrolytic water molecule, but it does not interact with the essential amino acids and has a high temperature factor (40.3 Å 2 ).
As found in all penicilloyl-serine transferases possessing a lysine residue in the conserved (Lys/His/Arg)-(Thr/Ser)-Gly triad, a water molecule (Wat 303 in the K15 enzyme) is located at the top of the catalytic pocket and plays a structural role in stabilizing the Lys 213 side chain. One may note that the Streptomyces R61 DD-carboxypeptidase is the only known enzyme of the family that has a histidine residue instead of a lysine or arginine residue in this triad.
Accessible Surface Area-The accessible surface area of the K15 enzyme crystal structure has a value of 10,960 Å 2 as computed with the DSSP program (36), and its distribution along the polypeptide chain is quite similar to that observed in the class A ␤-lactamases (38). The central helix ␣2 and the five-stranded ␤-sheet are inaccessible to solvent, except strand ␤2, which is more exposed. Helices ␣4 and ␣5 are also buried, and the other ␣-helices have an amphiphilic profile. Strands ␤2a and ␤2d of the small ␤-sheet are very well exposed, strand ␤2c is amphiphilic, and strand ␤2b is more buried. The more accessible regions (Val 1 -Lys 3 , Asn 50 -Leu 51 , Asn 70 -Asn 71 , Ile 145 -Ala 149 , and Gly 231 -Gly 232 ) correspond to the highest temperature factor values (Ͼ30 Å 2 ).
The analysis also reveals very low accessibility values for several charged residues of the K15 enzyme. Among these, Lys 38 and Lys 213 are directly involved in the dense hydrogen bond network of the catalytic cleft. Asp 99 interacts with the ␣-domain and probably stabilizes the position of the Ser 96 -Gly 97 -Cys 98 motif, as does Asp 131 in the class A ␤-lactamases (39). Asp 143 and Asn 150 , at the bottom of the active pocket, interact with protein core atoms, allowing the positioning of the carbonyl group of Asp 143 in the catalytic cleft. Arg 248 , near strand ␤3, may play the same role as Arg 220 or Arg 244 in the class A ␤-lactamases (40). Two polar residues, Asn 198 and Asn 200 , above the catalytic pocket, are also quite buried. They may be compared with the Asp 214 (Asn 214 ) /Asp 233 pair in class A ␤-lactamases and the Glu 272 /His 234 pair in class C ␤-lactamases. These latter residues are involved in specific interactions, in particular with a water molecule equivalent to Wat 303 in the K15 enzyme (16). DISCUSSION Several scenari may be evoked through which the present day penicilloyl-serine transferases of different groups and classes have evolved from a protein ancestor similar to the K15 enzyme in structure and catalytic properties. One evolution pathway is loss of peptidase activity, loss of membrane association site, and acquired catalyzed hydrolysis of the penicilloyl enzyme. The resulting exocellular ␤-lactamases hydrolyze penicillin into biologically inactive metabolites, providing the bacteria with a defensive mechanism of remarkable efficacy. On good ␤-lactam substrates, the TEM ␤-lactamase may turn over 1000 times/s or more. In this respect and in comparison with the K15 enzyme, the TEM ␤-lactamase has a 10 6 -fold or more increased catalytic center activity. In structural terms, the class A ␤-lactamases have lost the four-stranded ␤-sheet at the surface of the all-␣-domain; they have acquired an additional ␣-helix at the amino-terminal end of the polypeptide chain and a specifically designed ⍀-loop at the entry of the active site. Essentially, however, the overall polypeptide fold has been maintained.
Another possible evolutionary pathway that retains the activity toward peptide substrates would be the acquisition of membrane-associated carboxyl-terminal amphiphilic ␣-helices, giving rise to the membrane-bound monofunctional DD-carboxypeptidases/PBPs. Finally, fusion of the acylserine transferase polypeptide to the carboxyl-terminal end of other polypeptide chains may have given rise to the membranebound multimodular PBPs. The acylserine transferase/penicillin-binding module of PBP2x of S. pneumoniae (Protein Data Bank code 1PMD) has the signature fold topology and active site of the K15 enzyme. But it also possesses two inserts that form an additional groove at the surface of the module, and the head of the non-penicillin-binding module, which is shaped like a pair of sugar tongs, fits into the non-catalytic groove of the acylserine transferase (20).
The structurally related K15 DD-transpeptidase/PBP, DD-carboxypeptidases/PBPs, and serine ␤-lactamases fulfill different functions and catalyze distinct reactions (albeit exhibiting a similar catalytic mechanism). They illustrate the concept according to which proteins unrelated in sequence and function may diverge from a common ancestor and new enzyme functions may evolve by local structural changes while retaining the same basic fold. The question of which structural features determine the different functionalities (peptide bond synthesis versus hydrolysis, penicillin binding versus hydrolysis) and vastly different kinetic properties of the monofunctional penicilloyl-serine transferases remains open.