The high resolution crystal structure for class A beta-lactamase PER-1 reveals the bases for its increase in breadth of activity.

The treatment of infectious diseases by beta-lactam antibiotics is continuously challenged by the emergence and dissemination of new beta-lactamases. In most cases, the cephalosporinase activity of class A enzymes results from a few mutations in the TEM and SHV penicillinases. The PER-1 beta-lactamase was characterized as a class A enzyme displaying a cephalosporinase activity. This activity was, however, insensitive to the mutations of residues known to be critical for providing extended substrate profiles to TEM and SHV. The x-ray structure of the protein, solved at 1.9-A resolution, reveals that two of the most conserved features in class A beta-lactamases are not present in this enzyme: the fold of the Omega-loop and the cis conformation of the peptide bond between residues 166 and 167. The new fold of the Omega-loop and the insertion of four residues at the edge of strand S3 generate a broad cavity that may easily accommodate the bulky substituents of cephalosporin substrates. The trans conformation of the 166-167 bond is related to the presence of an aspartic acid at position 136. Selection of class A enzymes based on the occurrence of both Asp(136) and Asn(179) identifies a subgroup of enzymes with high sequence homology.


INTRODUCTION
Pseudomonas aeruginosa, a prevalent micro-organism responsible for several infections in human, is intrinsically resistant to many antibiotics (1). This resistance simultaneously arises from the low permeability of its outer membrane (2), the existence of an active efflux system (3,4), and the production of a chromosomally encoded inducible class C β-lactamase (5). This bacterium reinforces its resistance to β-lactam antibiotics by expressing a chromosomally encoded class A cephalosporinase, PER-1 (for Pseudomonas Extended Resistance).
PER-1 was first detected in 1993 in a strain isolated from a Turkish patient (6). It was subsequently identified in nosocomial strains of Salmonella typhimurium and Acinetobacter baumanii in Turkey (7,8) and more recently in France (9). PER-2, a closely related enzyme sharing 86% homology, has been found in South America (10). These enzymes hydrolyze efficiently penicillins and cephalosporins, but not cephamycins or carbapenems, and are susceptible to clavulanic acid inhibition (6,10). PER enzymes belong to the class A β-lactamases (10)(11)(12) but the sequence identity with the TEM and SHV enzymes is only 27% (11). It was proposed that PER enzymes and various β-lactamases from Bacteroides species (13-15) may constitute a subgroup of the class A enzymes (11).
Class A β-lactamases hydrolyze β-lactam antibiotics through a double displacement mechanism with a transient acylation of the catalytic Ser70 residue. Eight X-ray structures of apo-enzymes, TEM-1 and TOHO-1 from Escherichia coli (16,17), PC1 from Staphylococcus aureus (18), SHV from Klebsiella pneumoniae (19), NMC-A from Enterobacter cloacae (20), MFO from Mycobacterium fortuitum (PDB entry: 1MFO), BLIC from Bacillus licheniformis (21), and SAG from Streptomyces albus G (22) have been solved. These enzymes display a very similar fold and the detailed comparisons of the structures were helpful in relating some by guest on July 25, 2018 http://www.jbc.org/ Downloaded from 5 significant differences in substrate profile to local structural features (23,24). The structural impact of point mutations leading to some 100 extended spectrum enzymes in the TEM and SHV families is less documented than the possible function of the invariant residues of the catalytic machinery (25)(26)(27)(28). Several studies supported the hypothesis that the cephalosporinase activity of these extended substrate enzymes was related to an increased flexibility of the Ω-loop region and to alterations of the S3 strand, two of the regions lining the active site (24,(29)(30)(31)(32)(33).
Surprisingly, site-directed mutagenesis on the PER-1 enzyme showed that none of the residues responsible for the cephalosporinase activity in the TEM and SHV families (104, 164, 179, 238, and 240) were implicated in the substrate profile of this enzyme (34,35). The 1.9 Å Xray structure of PER-1 presented in this report reveals a completely new fold of the Ω-loop region, so far one of the most conserved feature in the class A enzymes. Structure and sequence analysis suggest that PER-1 defines a group of class A β-lactamases which can be recognized by the presence of an aspartic acid residue at position 136. The insertion of four amino acids in the 238-242 region, together with the Ω-loop fold, lead to a significant increase in size of the substrate binding pocket.

Data collection and phasing
Diffracted intensities were measured on the W32 beam line at LURE (Orsay, France) on a large MAR Research imaging plate. The data of the native and of the first HgCl 2 derivative (HgCl 2 1) were collected at 20°C. This derivative was obtained by soaking the crystal for 64 h in a capillary at a final concentration of 10 mM. The data for the second HgCl 2 derivative (HgCl 2 2) were collected at -160°C. Soaking for the HgCl 2 2 derivative was performed during 69 h in a drop of mother liquor at a final concentration of 5 mM. For cryocooling, the crystal was immersed for 10 seconds in the reservoir solution complemented with 20% ethylene-glycol (w/v) prior to flash-cooling in a stream of nitrogen gas. 9

Structure determination
The quality of the SIRAS electron density map computed at 2.5 Å resolution, using the two data sets of the mercury derivative for phasing, allowed an unambiguous main chain tracing except for residues 104-109, 162-164 and 170-176. One half of the side chain were built at that stage. Inclusion of the high resolution data followed by a few rounds of refinement led to R and R free values of 0.14 and 0.18, respectively at 1.9 Å resolution. The refined structure of PER-1 includes 274 residues and 155 water molecules. The first N-terminal (Gln25) and the last three Cterminal residues (Ser298, Pro299, and Asn300) have no electron density. The average temperature factors was 20.17 Å 2 , close to the value estimated from the Wilson plot (16.9 Å 2 ) (48). Coordinates error was evaluated to be 0.16 Å from a Luzzati plot (49).

Overall structure and catalytic machinery
The PER-1 β-lactamase (274 residues) is made of two domains. The first one (residues 26-62 and 218-297) folds as a five stranded anti-parallel β-sheet with both N-and C-terminal αhelices packed on one side of the sheet. The second domain (residues 69-215) is mainly built of α-helices and loops. The active site is defined by the interface between the two domains ( Figure   1).
The side chains of the essential catalytic residues, Ser70, Lys73, Ser130, and Glu166, the atoms forming the oxyanion hole (the main chain nitrogen atoms of Ser70 and Thr237) and the deacylating water molecule ( Figure 2) are found in identical relative positions compared to other class A β-lactamases (rmsd = 0.350 ± 0.110 Å) , suggesting a conserved catalytic mechanism. A water molecule occupies the oxyanion hole, at 2.8 and 3.2 Å of Thr237N and Ser70N, respectively. A sulfate anion, likely provided by the crystallization medium, is found at hydrogen by guest on July 25, 2018 http://www.jbc.org/ Downloaded from bond distance to Ser130, Thr235 and Thr236. A sulfate ion bound in a similar position was also observed in the TEM structure (16).

Insertions
Sequence alignment of PER-1 with the class A β-lactamases pointed out the occurrence of three insertions (11,34). Two of them (Q103a and N103b, Q112a and G112b ) are located in the loop connecting helices H2 and H4 ( Figure 1). They likely contribute to the different conformation of this region when compared to that of the TEM enzyme (rmsd on common main chain atoms is 2.6 Å) ( Figure 3). This conformation nevertheless preserves the Cα positions of residues 104 and 105, two residues known to be important in class A enzymes for substrate binding and catalysis (50,51). In PER-1, the γ-hydroxyl group of the Thr104 side chain is hydrogen bonded (2.9 Å) to the side chain of the invariant Asn132 residue (Figures 3 and 4). This conformation of residue104 has never been observed in class A enzymes where the polar interaction to Asn132 was always provided by the main chain oxygen atom of residue 104 ( Figure 3). Trp105 (an aromatic residue in most class A enzymes, Figure 4) has the same location as Tyr105 in TEM. In this protein, the aromatic ring is at van der Waals distance to the bound substrate in the crystal structures of enzyme-ligand complexes (52)(53)(54).
The third insertion occurs in a region where the sequence and the local fold were related to the extension of substrate profile in the TEM and SHV β-lactamases (28,32,33). In PER-1, four amino acids are inserted after residue 240 (Lys240a, Ala240b, Gly240c, and Lys240d). The loop 238-242 that connects strands S3 and S4 protrudes in the solvent, away from the Ω-loop region ( Figure 1). As a consequence, the adjacent loop between strand S5 to helix H11 is short, and helix H11 starts at residue 272. It could be noticed that the start of helix H11 (at position 272 or 276) and therefore the length of the S5-H11 connection (long or short, respectively) partition the class A enzymes into two groups. It would seem from structures examination that the long connection, when it occurs, shields from solvent the aromatic residue found at position 241 (TOHO-1, SAG, MFO, BLIC, PC1, NMCA) ( Figure 4).
The Ω Ω Ω Ω-loop fold The Ω-loop is an idiosyncrasy of class A β-lactamases. Its fold and the cis peptide bond between residues 166 and 167 were typical and invariant features observed in the 8 structures The Ω-loop region in PER-1 is highly organized in secondary structure elements. The  (16)(17)(18)(19)(20)22,55) where the invariant interaction between Arg164 and Asp179 is directly implicated in the substrate profile of these enzymes ( Figure 5).
The fold of the Ω-loop in PER-1 maintains Glu166 in the right location for the catalytic function and for binding the deacylating water molecule. This fold however brings His170 8 Å away from the position of the homologous residue in the other class A enzymes. In these structures, Asn170, a highly conserved amino acid in this family ( Figure 4) is the second ligand of the deacylating water molecule. In PER-1, this function is achieved by Gln69 (Figure 2).

DISCUSSION
Although PER-1 is a typical class A enzyme with respect to the conservation of the catalytic machinery, its three dimensional structure revealed that this super-family is not as homogeneous in structure as it was thought. It was unexpected to find a new fold of the Ω-loop, a region considered to be a canonical motif in these enzymes. This finding suggested to perform a selection of the β-lactamases only based on the occurrence of an aspartic acid at position 136.