Structural and Mutagenic Analysis of Metallo-β-Lactamase IMP-18

Takamitsu Furuyama; Haruka Nonomura; Yoshikazu Ishii; Nancy D Hanson; Akiko Shimizu-Ibuka

doi:10.1128/AAC.00985-16

. 2016 Aug 22;60(9):5521–5526. doi: 10.1128/AAC.00985-16

Structural and Mutagenic Analysis of Metallo-β-Lactamase IMP-18

Takamitsu Furuyama ^a, Haruka Nonomura ^b, Yoshikazu Ishii ^c, Nancy D Hanson ^d, Akiko Shimizu-Ibuka ^a,^b,^✉

PMCID: PMC4997881 PMID: 27381398

Abstract

IMP-type metallo-β-lactamases (MBLs) are exogenous zinc metalloenzymes that hydrolyze a broad range of β-lactams, including carbapenems. Here we report the crystal structure of IMP-18, an MBL cloned from Pseudomonas aeruginosa, at 2.0-Å resolution. The overall structure of IMP-18 resembles that of IMP-1, with an αβ/βα “folded sandwich” configuration, but the loop that covers the active site has a distinct conformation. The relationship between IMP-18's loop conformation and its kinetic properties was investigated by replacing the amino acid residues that can affect the loop conformation (Lys44, Thr50, and Ile69) in IMP-18 with those occupying the corresponding positions in the well-described enzyme IMP-1. The replacement of Thr50 with Pro considerably modified IMP-18's kinetic properties, specifically those pertaining to meropenem, with the k_cat/K_m value increased by an order of magnitude. The results indicate that this is a key residue that defines the kinetic properties of IMP-type β-lactamases.

INTRODUCTION

The discovery and development of antibiotics, such as β-lactams, aminoglycosides, and quinolones, have provided effective tools for the treatment of infectious diseases caused by bacteria. However, their therapeutic use rapidly led to the emergence of antibiotic-resistant bacteria, threatening their clinical efficacy (1). Bacteria developed several strategies to escape these lethal molecules, such as the synthesis of β-lactamases to hydrolyze β-lactam antibiotics, decreased target sensitivity, porin mutations that decrease membrane permeability, and/or the efflux system modification (1 – 3). The production of β-lactamases is the main defense mechanism against β-lactam-based antibiotics, especially for Gram-negative bacteria (4).

β-Lactamases are classified into four groups (A to D). Class B β-lactamases, also known as metallo-β-lactamases (MBLs), require a zinc ion(s) for their catalytic activity and generally exhibit a high hydrolytic activity toward carbapenems. Furthermore, they are not affected by the commercially available β-lactamase inhibitors (5). MBLs are further divided into three subclasses (B1, B2, and B3) based on sequence similarities and structural features (6, 7). Subclass B1 includes the transferable MBLs, such as IMP, VIM, GIM, and NDM. Bacteria with IMP-type enzymes have spread throughout the world, and the IMP group now has more than 50 variants (http://www.laced.uni-stuttgart.de). These enzymes possess a broad substrate specificity and a high affinity for cephalosporins and carbapenems but a low activity toward temocillin (8).

IMP-18 shares 80% amino acid identity with IMP-1, a well-studied IMP-type enzyme in terms of kinetic and structural properties. Kinetic evaluations of IMP-18 revealed that the overall turnover rates are lower than those for other IMP-type variants, especially toward meropenem (9). In order to investigate the structural basis for the substrate specificity of IMP-type enzymes, we solved the crystal structure of IMP-18 and performed a kinetic analysis of several IMP-18 mutants. The mutants generated in this study modified the residues of IMP-18 determined by the crystal structure to have the largest impacts. These residues were replaced with those found in IMP-1, and the kinetic properties of the mutants were evaluated.

MATERIALS AND METHODS

X-ray data collection and structure determination for wild-type IMP-18.

The protocols for overexpression and purification of IMP-18 were described in our previous report (10). We optimized the crystallization conditions as follows, based on the results of our previous screening (10), to obtain crystals suitable for data collection: 0.1 M sodium citrate buffer (pH 5.2), 20% (wt/vol) polyethylene glycol 4000, 3% (vol/vol) ethylene glycol, and 0.01 M strontium chloride (SrCl₂) at 283 K. The X-ray data were collected at beamlines BL5A, NW12A, and NE3A at the Photon Factory, KEK (Tsukuba, Japan). The diffraction patterns were indexed, integrated, and scaled using HKL-2000 (11) or iMosflm (12), followed by the programs of the CCP4 suite (13). The search model was generated using SWISS-MODEL (14), based on the amino acid sequence of IMP-18 and the structure of IMP-1 (PDB entry 1DDK) (15). The model was subjected to molecular replacement with MOLREP (16). The model was built using COOT (17) and refined using Refmac (18). The stereochemical quality of the generated model was validated using RAMPAGE (19).

Preparation of IMP-18 mutants.

The IMP-18 mutants were constructed by site-directed mutagenesis with a PrimeSTAR Mutagenesis Basal kit (TaKaRa Bio Co., Japan). The pET28a-imp18 plasmid, constructed for the expression of wild-type IMP-18 (10), was used as a template for the construction of K44N, T50P, and I69F single mutants. The oligonucleotide primers imp18-K44N-for (5′-GAA GTT AAC GGT TGG GGT GTA GTC ACA-3′) and imp18-K44N-rev (5′-CCA ACC GTT AAC TTC TTC AAA CGA AGT-3′) were synthesized for the K44N mutation, imp18-T50P-for (5′-GTG TGG TAC CGA AAC ACG GTT TAG TGG TT-3′) and imp18-T50P-rev (5′-GTT TCG GTA CCA CAC CCC AAC CTT TAA CT-3′) for the T50P mutation, and imp18-I69F-for (5′-CCA TTT ACC GCG AAA GAT ACT GAA AAA TTA-3′) and imp18-I69F-rev (5′-TTT CGC GGT AAA TGG AGT ATC TAT CAG ATA-3′) for the I69F mutation. The expression plasmids for the K44N, T50P, and I69F mutants were designated pET28a-imp18K44N, pET28a-imp18T50P, and pET28a-imp18I69F, respectively. For the K44N/T50P mutant, pET28a-imp18T50P was used as the template, and the primers for site-directed mutagenesis were imp18K44NinT50Pfor (5′-AAG TTA ACG GTT GGG GTG TAG TCC CG-3′), and imp18K44NinT50Prev (5′-CCC AAC CGT TAA CTT CTT CAA ACG AA-3′). The nucleotide sequence of the mutated region in each expression plasmid was confirmed by DNA sequencing performed using an ABI Prism 3100 genetic analyzer (Applied Biosystems, Waltham, MA). All the mutants were expressed and purified according to the protocols used for the wild-type enzyme (10) and then subjected to kinetic analysis.

Kinetic assays.

The following antibiotics and chemicals were used for the kinetic analysis. Ampicillin (Δε₂₃₅ = −820 M⁻¹ cm⁻¹) and cefotaxime (Δε₂₆₀ = −7,500 M⁻¹ cm⁻¹) were purchased from Wako Chemicals (Tokyo, Japan). Benzylpenicillin (Δε₂₃₅ = −775 M⁻¹ cm⁻¹), cephaloridine (Δε₂₆₀ = −10,000 M⁻¹ cm⁻¹), ceftazidime (Δε₂₆₀ = −9,000 M⁻¹ cm⁻¹), imipenem (Δε₃₀₀ = −9,000 M⁻¹ cm⁻¹), and meropenem (Δε₃₀₀ = −6,500 M⁻¹ cm⁻¹) were purchased from Sigma-Aldrich (St. Louis, MO). Nitrocefin (Δε₄₈₂ = 15,000 M⁻¹ cm⁻¹) was purchased from Unipath Oxoid (Basingstoke, United Kingdom). The kinetic analysis was carried out as reported by Borgianni et al. (9). The reaction was performed at 30°C in 500 μl of 10 mM HEPES buffer (pH 7.5) containing 50 μM ZnSO₄, and all measurements were made using a Jasco V-530 spectrophotometer. The enzyme was diluted by adding bovine serum albumin (BSA) to the buffer, to a final concentration of 20 μg ml⁻¹, to prevent denaturation. At least three independent progress curves were obtained for each substrate, until reproducible results were obtained.

Accession number(s).

The coordinate and structure factor files from this study have been deposited in the Protein Data Bank under accession code 5B3R.

RESULTS

Overall structure of IMP-18 β-lactamase.

The structure of IMP-18 was determined by molecular replacement, using IMP-1 as a template for the starting model, and then refined to 2.0 Å (Table 1). The structure of IMP-18 contains two molecules (A and B) in an asymmetric unit. Molecule A includes residues 22 to 41 and 45 to 238, with an average temperature factor of 30.07 Å². Molecule B consists of residues 21 to 239, with an average temperature factor (28.00 Å²) that is lower than that for molecule A. The overall root mean square deviation (RMSD) for C-α between molecules A and B is 0.27 Å. The C-α RMSD is the largest for the loop region extending from Glu42 to Val48 (residues 60 to 66 in the MBL standard numbering scheme [6, 7]; the standard numbers are given in parentheses throughout this paper), which connects two antiparallel β-strands and is termed the “flap” or “loop L3” typical of class B1 MBLs. Symmetry operations revealed that this loop is involved in crystal packing in molecule B because it penetrates deep into the active site groove of the equivalent molecule (Fig. 1A). Molecule A also interacts with its equivalent molecule in crystal packing, but the interaction is not exactly the same as that seen in molecule B, since the loop region is partly disordered in molecule A (Fig. 1A). The active site contains two zinc ions (Zn1 and Zn2), as reported for other MBLs, with a citrate molecule in close vicinity (Fig. 1B). The electron density of Zn2 is lower than that of Zn1, with the occupancy being set to 0.5 in both molecules A and B.

TABLE 1.

Data collection and structure refinement

Parameter	Value or description
Data collection statistics
Unit cell dimensions (a, b, c)	78.55, 87.38, 105.75
Space group	P222₁
Resolution (Å)	36.82–1.97 (2.08–1.97)
No. of observed reflections	728,578 (97,345)
No. of unique reflections	50,965 (7,079)
Completeness	98.7 (95.4)
Multiplicity	14.3 (13.8)
I/σ(I)	6.6 (4.2)
R_sym	0.073 (0.176)
Refinement statistics
Resolution (Å)	30.00–2.00 (2.05–2.00)
No. of reflections	46,813 (3,324)
No. of atoms	3,576
Protein atoms	3,384
Nonprotein atoms	192
R_work	0.201 (0.232)
R_free	0.232 (0.246)
RMSD from ideal
Bond lengths (Å)	0.0080
Bond angles (°)	1.3173
Avg B factor (Å²)	29.00
Protein (chain A) atoms	30.07
Protein (chain B) atoms	28.00
Nonprotein atoms	30.89

Open in a new tab

FIG 1 — Structural features of IMP-18. (A) Interaction between molecule B and its equivalent during crystal packing. The residues participating in the interaction are labeled in the molecule colored green. Molecule A and its equivalent are colored gray. (B) The two zinc ions (Zn1 and Zn2) and the citrate molecule observed in the active site.

Structural comparison of IMP-18 and other IMP-type enzymes.

Superpositioning of IMP-18 (molecule B) and IMP-1 (PDB entry 1DDK) yielded an RMSD of 0.64 Å for 215 C-α atoms, and that of IMP-18 and IMP-2 (PDB entry 4UBQ) yielded an RMSD of 0.63 Å for 216 C-α atoms (Fig. 2A). The largest structural difference was observed for the residues extending from Phe40 to Thr50 (residues 58 to 68 in the standard numbering scheme). This region resides near the active site and includes the flap region. The distance between Trp46 C-α of IMP-18 and the corresponding atom of IMP-1 (Trp28 C-α) is 7.3 Å. For IMP-1, this loop was reported to adopt an “open” conformation, and it becomes “closed” when a substrate/inhibitor is bound to the active site (20). A structural comparison of IMP-18, free IMP-1, and IMP-1 complexed with a mercaptocarboxylate inhibitor (PDB entry 1DD6) showed that the loop of IMP-18 is not “closed” as observed with the IMP-1–inhibitor complex. Compared to the IMP-1 loop, the IMP-18 loop is sliding sideways in a different direction.

FIG 2 — Comparison of flap loop conformations of IMP-18 and IMP-type enzymes. (A) Crystal structure of IMP-18 (green) superimposed on those of free IMP-1 (PDB entry 1DDK; violet), IMP-1 complexed with a mercaptocarboxylate inhibitor (PDB entry 1DD6; molecule A, pink; molecule B, blue), and IMP-2 (PDB entry 4UBQ; orange). (B) Partial alignment of sequences of IMP-type enzymes. The numbered residues mentioned in the text are indicated by arrows. The secondary structure of IMP-18 is shown above the alignments, with the β-strands shown as white arrows, an α-helix as a white rectangle, and the loops as black bars.

Kinetic properties of IMP-18 mutants.

Given the significant difference in flap loop conformation observed between IMP-18 and IMP-1, we aligned their amino acid sequences to find that only a few amino acids differ in this region between these enzymes (Fig. 2B). We focused on three residues: Lys44 (residue 61), positioned on this loop; Thr50 (residue 68), positioned on the C-terminal side of the loop; and the well-conserved Phe residue at position 69 (residue 87) in IMP-type enzymes. However, the latter is replaced with Ile in IMP-18, and the side chain faces the β-strand positioned on the N-terminal side of the loop (Fig. 2A).

The roles of these residues were investigated by kinetic analysis of four mutants: the K44N, T50P, K44N/T50P, and I69F mutants. The kinetic parameters for penicillins and carbapenems are summarized in Table 2, and those for cephalosporins are shown in Table 3. Each mutant exhibited distinct kinetic properties. For the K44N mutant, the k_cat and k_cat/K_m values tended to be higher than those of the wild-type enzyme, but the impact of this mutation was rather moderate. Regarding the T50P mutant, the impact of the substitution was considerably more apparent. The k_cat values were higher than those of the wild-type enzyme for all the tested substrates, especially for cephalosporins and carbapenems. The catalytic efficiency of this mutant was higher for cephaloridine, cefotaxime, and nitrocefin, whereas the k_cat/K_m value was lower for penicillins and ceftazidime because of the 2- to 4-fold increase in K_m values. The most significant change was observed in the parameters for meropenem. The k_cat value of the T50P mutant (1.28 s⁻¹) was 13-fold higher than that of the wild-type enzyme (0.0984 s⁻¹), and the K_m value was 2-fold lower (6.4 μM for the T50P mutant and 13.1 μM for the wild-type enzyme), together resulting in a substantial increase in the k_cat/K_m value, by an order of magnitude (0.230 s⁻¹ μM⁻¹ for the T50P mutant and 0.00757 s⁻¹ μM⁻¹ for the wild-type enzyme). The K44N/T50P mutant exhibited an even more significant increase in hydrolytic activity. For this double mutant, the k_cat values were higher than those of the T50P mutant for all tested substrates. Compared to those of the wild-type enzyme, the k_cat/K_m values were 2 to 3 times higher for cephaloridine, cefotaxime, nitrocefin, and imipenem and approximately 40 times higher for meropenem (0.325 s⁻¹ μM⁻¹).

TABLE 2.

Kinetic parameters of wild-type and mutant IMP-18 β-lactamases for penicillins and carbapenems

β-Lactamase and parameter	Value for antibiotic
β-Lactamase and parameter	Ampicillin	Penicillin G	Imipenem	Meropenem
Wild type
k_cat (s⁻¹)	33.5 ± 3.4	49.3 ± 1.1	22.9 ± 1.3	0.0984 ± 0.0041
K_m (μM)	70.8 ± 7.3	48.6 ± 4.5	9.43 ± 0.42	13.1 ± 1.2
k_cat/K_m (s⁻¹ μM⁻¹)	0.474 ± 0.038	1.02 ± 0.09	2.44 ± 0.17	0.00759 ± 0.00076
K44N mutant
k_cat (s⁻¹)	48.2 ± 4.1	70.3 ± 5.3	30.0 ± 1.6	0.114 ± 0.005
K_m (μM)	71.2 ± 6.8	44.1 ± 3.0	9.13 ± 1.2	14.8 ± 1.6
k_cat/K_m (s⁻¹ μM⁻¹)	0.677 ± 0.018	1.60 ± 0.17	3.34 ± 0.46	0.00779 ± 0.00079
T50P mutant
k_cat (s⁻¹)	45.4 ± 5.4	63.1 ± 6.3	62.1 ± 7.3	1.28 ± 0.14
K_m (μM)	212 ± 36	184 ± 27	24.1 ± 2.2	6.4 ± 2.7
k_cat/K_m (s⁻¹ μM⁻¹)	0.217 ± 0.017	0.345 ± 0.024	2.58 ± 0.23	0.230 ± 0.081
K44N/T50P mutant
k_cat (s⁻¹)	92.6 ± 8.8	126 ± 12	115 ± 10	1.99 ± 0.20
K_m (μM)	204 ± 21	242 ± 23	20.4 ± 3.0	6.29 ± 1.09
k_cat/K_m (s⁻¹ μM⁻¹)	0.458 ± 0.058	0.524 ± 0.058	5.76 ± 0.92	0.325 ± 0.067
I69F mutant
k_cat (s⁻¹)	16.4 ± 1.7	99.7 ± 5.3	19.8 ± 1.4	0.0972 ± 0.0051
K_m (μM)	7.60 ± 0.61	14.5 ± 1.2	6.17 ± 0.43	8.66 ± 0.47
k_cat/K_m (s⁻¹ μM⁻¹)	2.18 ± 0.32	6.92 ± 0.60	3.22 ± 0.23	0.0112 ± 0.0005

Open in a new tab

TABLE 3.

Kinetic parameters of wild-type and mutant IMP-18 β-lactamases for cephalosporins

β-Lactamase and parameter	Value for antibiotic
β-Lactamase and parameter	Cephaloridine	Ceftazidime	Cefotaxime	Nitrocefin
Wild type
k_cat (s⁻¹)	0.465 ± 0.027	1.34 ± 0.10	0.731 ± 0.041	83.4 ± 15.1
K_m (μM)	0.973 ± 0.171	3.02 ± 0.28	0.918 ± 0.033	19.9 ± 2.3
k_cat/K_m (s⁻¹ μM⁻¹)	0.490 ± 0.067	0.445 ± 0.046	0.796 ± 0.032	4.19 ± 0.57
K44N mutant
k_cat (s⁻¹)	0.533 ± 0.049	1.66 ± 0.09	0.767 ± 0.042	118 ± 8
K_m (μM)	0.477 ± 0.053	2.66 ± 0.29	0.738 ± 0.099	25.5 ± 3.1
k_cat/K_m (s⁻¹ μM⁻¹)	1.13 ± 0.14	0.629 ± 0.057	1.06 ± 0.18	4.66 ± 0.37
T50P mutant
k_cat (s⁻¹)	1.30 ± 0.09	3.82 ± 1.43	2.55 ± 0.36	138 ± 25
K_m (μM)	0.766 ± 0.066	12.3 ± 1.0	1.67 ± 0.20	18.7 ± 1.3
k_cat/K_m (s⁻¹ μM⁻¹)	1.71 ± 0.21	0.316 ± 0.119	1.54 ± 0.24	7.43 ± 1.53
K44N/T50P mutant
k_cat (s⁻¹)	2.27 ± 0.10	7.33 ± 0.45	3.62 ± 0.35	215 ± 24
K_m (μM)	0.497 ± 0.067	13.7 ± 0.8	1.88 ± 0.35	17.9 ± 2.0
k_cat/K_m (s⁻¹ μM⁻¹)	4.62 ± 0.43	0.534 ± 0.028	2.02 ± 0.53	12.2 ± 2.1
I69F mutant
k_cat (s⁻¹)	0.511 ± 0.062	4.56 ± 0.20	0.820 ± 0.059	172 ± 29
K_m (μM)	0.419 ± 0.054	2.40 ± 0.33	0.742 ± 0.050	33.0 ± 4.1
k_cat/K_m (s⁻¹ μM⁻¹)	1.25 ± 0.29	1.93 ± 0.24	1.11 ± 0.12	5.20 ± 0.61

Open in a new tab

The kinetic properties of the I69F mutant were distinct from those of the other mutants. This mutant exhibited few changes compared to the wild-type enzyme with respect to the carbapenem hydrolysis parameters. As a whole, a decrease in K_m was observed for most of the tested substrates compared to the values for the wild-type enzyme. The substantial decrease in K_m for penicillins, accompanied by an increase in k_cat values for penicillin G, ceftazidime, and nitrocefin, resulted in higher k_cat/K_m values for all the tested substrates, particularly for penicillins. The k_cat/K_m values for ampicillin, penicillin G, and ceftazidime were higher than those of the wild-type enzyme by 5-fold, 7-fold, and 4-fold, respectively.

Impacts of mutations on substrate binding and hydrolysis.

To understand how the mutations affected substrate binding/recognition, we superimposed the structures of IMP-18 and NDM-1 complexed with hydrolyzed benzylpenicillin or meropenem (PDB entries 4EYF and 4EYL) (Fig. 3). A carboxyl group of the citrate bound to IMP-18 is in the position of the carboxylate of the five-membered ring. Another carboxyl group of the citrate points toward Zn1 and is positioned close to the substrate carboxylate derived from the lactam carbonyl. Compared to that of IMP-1, the flap loop of IMP-18 is sliding over the R1 group of the substrates. The side chain of Ile69 resides near the R1 group of benzylpenicillin (Fig. 3).

FIG 3 — Superpositioning of IMP-18 (green), free IMP-1 (violet), NDM-1 complexed with hydrolyzed benzylpenicillin (PDB entry 4EYF; orange), and NDM-1 complexed with hydrolyzed meropenem (PDB entry 4EYL; cyan).

DISCUSSION

The crystallographic analysis clarified the structural features of IMP-18. IMP-18 adopts an αβ/βα sandwich fold, and the active site is located at the interface between the domains, which is typical for MBLs (21). Symmetry operations showed that the flap loop of one molecule is inserted into the active site groove of the equivalent molecule, implying that the conformation of the flap is affected by this symmetric contact. Two zinc ions (Zn1 and Zn2) and a citrate molecule are observed in the active site. The low electron density of Zn2 suggests a lower affinity for the binding site than that of Zn1. In this crystal structure, Zn2 may be partially displaced by citric acid, which can function as a chelating agent for Zn ions (22).

Superpositioning of the structures of IMP-1 and IMP-18 revealed a significant difference in the flap region. Although an open, disordered conformation of the flap is found in many crystal structures of MBLs, the flap of IMP-1 becomes “closed” as a substrate/inhibitor binds to the enzyme (20). In the IMP-18 structure, however, the flap loop adopts a “closed” conformation in the absence of substrate/inhibitor, and this conformation is slightly different from the closed conformation of IMP-1 (Fig. 2A). Nuclear magnetic resonance (NMR) analysis of CcrA, an MBL from Bacteroides fragilis, clearly showed that this region is more flexible than the whole molecule and that inhibitor binding decreases its flexibility, suggesting that the residues forming this strand-loop-strand region participate in substrate binding (23). Furthermore, mutagenic analysis of the subclass B1 MBLs IMP-1 and BcII indicated that the loop affects ligand binding and the turnover rate (24). In the present study, we showed that the conformational features of the IMP-18 flap may influence the catalytic properties of this enzyme, though the flap conformation seems to be stabilized by crystal lattice contacts.

The kinetic analysis presented here supports the above-mentioned hypothesis. The replacement of Thr50 (residue 68) with Pro increased the turnover rate and/or catalytic efficiency for many substrates, especially for meropenem. Because it was previously reported that IMP-18 is characterized by lower k_cat values than those for IMP-1, especially toward meropenem (5 s⁻¹ for IMP-1 and 0.05 s⁻¹ for IMP-18) (9), we considered this mutation to have converted IMP-18 into an IMP-1-type enzyme. How can this point mutation change the kinetic properties so drastically? The side chain of the residue at position 50 (residue 68) is exposed to the solvent in the crystal structures of IMP-18 and other IMP-type enzymes. Thus, there should be no direct interaction between the side chain of this residue and the substrate. Alternatively, the introduction of a Pro residue may restrict and/or modify the loop conformation. There is a significant difference in the position of the flap loop relative to the substrate between IMP-18 and IMP-1 (Fig. 3). We suppose that the T50P mutation induces a change in flap conformation which moves it closer to that of IMP-1, a position likely more suitable for β-lactam hydrolysis. Although the T50P mutation considerably increased hydrolytic activity toward meropenem, it did not substantially change the activity toward another carbapenem, imipenem. The structural difference between imipenem and meropenem is the substituent attached to the five-membered thiazolidine ring. In meropenem, the substituent contains a large pyrrolidine ring, whereas imipenem has a long linear chain. Compared to imipenem, meropenem is a poor substrate of the wild-type IMP-18 enzyme, possibly because this pyrrolidine ring causes steric conflict with the flap loop of the enzyme. In this case, the change of the flap conformation caused by the T50P mutation may reduce the conflict, selectively increasing the hydrolytic activity toward meropenem. On the other hand, this conformational change may cause a loss of favorable interactions between the flap and the R1 group in penicillins, because of the increase in K_m and decrease in k_cat for penicillins observed with these mutants and the fact that the loop of IMP-18 is positioned closer to the R1 group of benzylpenicillin (Table 2; Fig. 3).

Structural comparison of IMP-18 and NDM-1 complexed with hydrolyzed substrates indicates that the side chain of Ile69 (residue 87) faces the R1 group of benzylpenicillin (Fig. 3) and thus may contribute to substrate binding as a constituent of the S1 hydrophobic pocket, as reported for IMP-1 (20). The kinetic analysis of the I69F mutant revealed lower K_m values for the tested substrates, except for nitrocefin, implying that this mutation enhances hydrophobic interactions between the aromatic ring of Phe and the R1 groups of the substrates. The results presented in this report are in agreement with those in a previous report by Materon et al. (25). They randomized 29 residues in or near the active site of IMP-1 to identify the residues critical for its function. The residues corresponding to those of IMP-18 mutated in our study were shown to be replaceable by other amino acids and not essential for the hydrolytic reaction of IMP-1. However, they also showed that the Phe at position 87 in IMP-1 is well conserved for ampicillin hydrolysis but tends to be replaced with Gly for cefotaxime hydrolysis (25).

In summary, the integration of crystallographic and kinetic analyses performed in this study revealed the structural features of IMP-18 and the roles of the residues in the flap region that are not conserved between IMP-18 and IMP-1. The residue at position 50 (residue 68) was identified as one of the key residues that define the kinetic properties of the IMP-type β-lactamases, and possibly the flap conformation.

ACKNOWLEDGMENTS

We thank Hiromi Yoshida and Takashi Tonozuka for their kind support and advice during data collection at KEK.

REFERENCES

1.Walsh C. 2000. Molecular mechanisms that confer antibacterial drug resistance. Nature 406:775–781. doi: 10.1038/35021219. [DOI] [PubMed] [Google Scholar]
2.Nikaido H, Pages JM. 2012. Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria. FEMS Microbiol Rev 36:340–363. doi: 10.1111/j.1574-6976.2011.00290.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Alekshun MN, Levy SB. 2007. Molecular mechanisms of antibacterial multidrug resistance. Cell 128:1037–1050. doi: 10.1016/j.cell.2007.03.004. [DOI] [PubMed] [Google Scholar]
4.Wilke MS, Lovering AL, Strynadka NC. 2005. β-Lactam antibiotic resistance: a current structural perspective. Curr Opin Microbiol 8:525–533. doi: 10.1016/j.mib.2005.08.016. [DOI] [PubMed] [Google Scholar]
5.Bebrone C. 2007. Metallo-β-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily. Biochem Pharmacol 74:1686–1701. doi: 10.1016/j.bcp.2007.05.021. [DOI] [PubMed] [Google Scholar]
6.Galleni M, Lamotte-Brasseur J, Rossolini GM, Spencer J, Dideberg O, Frere JM, Metallo-Beta-Lactamases Working Group. 2001. Standard numbering scheme for class B β-lactamases. Antimicrob Agents Chemother 45:660–663. doi: 10.1128/AAC.45.3.660-663.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Garau G, Garcia-Saez I, Bebrone C, Anne C, Mercuri P, Galleni M, Frere JM, Dideberg O. 2004. Update of the standard numbering scheme for class B β-lactamases. Antimicrob Agents Chemother 48:2347–2349. doi: 10.1128/AAC.48.7.2347-2349.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Cornaglia G, Giamarellou H, Rossolini GM. 2011. Metallo-β-lactamases: a last frontier for β-lactams? Lancet Infect Dis 11:381–393. doi: 10.1016/S1473-3099(11)70056-1. [DOI] [PubMed] [Google Scholar]
9.Borgianni L, Prandi S, Salden L, Santella G, Hanson ND, Rossolini GM, Docquier JD. 2011. Genetic context and biochemical characterization of the IMP-18 metallo-β-lactamase identified in a Pseudomonas aeruginosa isolate from the United States. Antimicrob Agents Chemother 55:140–145. doi: 10.1128/AAC.00858-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Furuyama T, Ishii Y, Ohya N, Tateda K, Hanson ND, Shimizu-Ibuka A. 2013. Purification, crystallization and preliminary X-ray analysis of IMP-18, a class B carbapenemase from Pseudomonas aeruginosa. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:1397–1400. doi: 10.1107/S1744309113030480. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Otwinowski Z, Minor W. 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276:307–326. doi: 10.1016/S0076-6879(97)76066-X. [DOI] [PubMed] [Google Scholar]
12.Battye TG, Kontogiannis L, Johnson O, Powell HR, Leslie AG. 2011. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D Biol Crystallogr 67:271–281. doi: 10.1107/S0907444910048675. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Collaborative Computational Project, Number 4. 1994. The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50:760–763. doi: 10.1107/S0907444994003112. [DOI] [PubMed] [Google Scholar]
14.Arnold K, Bordoli L, Kopp J, Schwede T. 2006. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201. doi: 10.1093/bioinformatics/bti770. [DOI] [PubMed] [Google Scholar]
15.Yamaguchi Y, Kuroki T, Yasuzawa H, Higashi T, Jin W, Kawanami A, Yamagata Y, Arakawa Y, Goto M, Kurosaki H. 2005. Probing the role of Asp-120(81) of metallo-β-lactamase (IMP-1) by site-directed mutagenesis, kinetic studies, and X-ray crystallography. J Biol Chem 280:20824–20832. doi: 10.1074/jbc.M414314200. [DOI] [PubMed] [Google Scholar]
16.Vagin A, Teplyakov A. 2010. Molecular replacement with MOLREP. Acta Crystallogr D Biol Crystallogr 66:22–25. doi: 10.1107/S0907444909042589. [DOI] [PubMed] [Google Scholar]
17.Emsley P, Lohkamp B, Scott WG, Cowtan K. 2010. Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66:486–501. doi: 10.1107/S0907444910007493. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Murshudov GN, Vagin AA, Dodson EJ. 1997. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53:240–255. doi: 10.1107/S0907444996012255. [DOI] [PubMed] [Google Scholar]
19.Lovell SC, Davis IW, Arendall WB III, de Bakker PI, Word JM, Prisant MG, Richardson JS, Richardson DC. 2003. Structure validation by Cα geometry: ϕ,ψ and Cβ deviation. Proteins 50:437–450. doi: 10.1002/prot.10286. [DOI] [PubMed] [Google Scholar]
20.Concha NO, Janson CA, Rowling P, Pearson S, Cheever CA, Clarke BP, Lewis C, Galleni M, Frere JM, Payne DJ, Bateson JH, Abdel-Meguid SS. 2000. Crystal structure of the IMP-1 metallo β-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor: binding determinants of a potent, broad-spectrum inhibitor. Biochemistry 39:4288–4298. doi: 10.1021/bi992569m. [DOI] [PubMed] [Google Scholar]
21.Palzkill T. 2013. Metallo-β-lactamase structure and function. Ann N Y Acad Sci 1277:91–104. doi: 10.1111/j.1749-6632.2012.06796.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Concha NO, Rasmussen BA, Bush K, Herzberg O. 1996. Crystal structure of the wide-spectrum binuclear zinc β-lactamase from Bacteroides fragilis. Structure 4:823–836. doi: 10.1016/S0969-2126(96)00089-5. [DOI] [PubMed] [Google Scholar]
23.Scrofani SD, Chung J, Huntley JJ, Benkovic SJ, Wright PE, Dyson HJ. 1999. NMR characterization of the metallo-β-lactamase from Bacteroides fragilis and its interaction with a tight-binding inhibitor: role of an active-site loop. Biochemistry 38:14507–14514. doi: 10.1021/bi990986t. [DOI] [PubMed] [Google Scholar]
24.Moali C, Anne C, Lamotte-Brasseur J, Groslambert S, Devreese B, Van Beeumen J, Galleni M, Frere JM. 2003. Analysis of the importance of the metallo-β-lactamase active site loop in substrate binding and catalysis. Chem Biol 10:319–329. doi: 10.1016/S1074-5521(03)00070-X. [DOI] [PubMed] [Google Scholar]
25.Materon IC, Beharry Z, Huang W, Perez C, Palzkill T. 2004. Analysis of the context dependent sequence requirements of active site residues in the metallo-β-lactamase IMP-1. J Mol Biol 344:653–663. doi: 10.1016/j.jmb.2004.09.074. [DOI] [PubMed] [Google Scholar]

[B1] 1.Walsh C. 2000. Molecular mechanisms that confer antibacterial drug resistance. Nature 406:775–781. doi: 10.1038/35021219. [DOI] [PubMed] [Google Scholar]

[B2] 2.Nikaido H, Pages JM. 2012. Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria. FEMS Microbiol Rev 36:340–363. doi: 10.1111/j.1574-6976.2011.00290.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3.Alekshun MN, Levy SB. 2007. Molecular mechanisms of antibacterial multidrug resistance. Cell 128:1037–1050. doi: 10.1016/j.cell.2007.03.004. [DOI] [PubMed] [Google Scholar]

[B4] 4.Wilke MS, Lovering AL, Strynadka NC. 2005. β-Lactam antibiotic resistance: a current structural perspective. Curr Opin Microbiol 8:525–533. doi: 10.1016/j.mib.2005.08.016. [DOI] [PubMed] [Google Scholar]

[B5] 5.Bebrone C. 2007. Metallo-β-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily. Biochem Pharmacol 74:1686–1701. doi: 10.1016/j.bcp.2007.05.021. [DOI] [PubMed] [Google Scholar]

[B6] 6.Galleni M, Lamotte-Brasseur J, Rossolini GM, Spencer J, Dideberg O, Frere JM, Metallo-Beta-Lactamases Working Group. 2001. Standard numbering scheme for class B β-lactamases. Antimicrob Agents Chemother 45:660–663. doi: 10.1128/AAC.45.3.660-663.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7.Garau G, Garcia-Saez I, Bebrone C, Anne C, Mercuri P, Galleni M, Frere JM, Dideberg O. 2004. Update of the standard numbering scheme for class B β-lactamases. Antimicrob Agents Chemother 48:2347–2349. doi: 10.1128/AAC.48.7.2347-2349.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8.Cornaglia G, Giamarellou H, Rossolini GM. 2011. Metallo-β-lactamases: a last frontier for β-lactams? Lancet Infect Dis 11:381–393. doi: 10.1016/S1473-3099(11)70056-1. [DOI] [PubMed] [Google Scholar]

[B9] 9.Borgianni L, Prandi S, Salden L, Santella G, Hanson ND, Rossolini GM, Docquier JD. 2011. Genetic context and biochemical characterization of the IMP-18 metallo-β-lactamase identified in a Pseudomonas aeruginosa isolate from the United States. Antimicrob Agents Chemother 55:140–145. doi: 10.1128/AAC.00858-10. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10.Furuyama T, Ishii Y, Ohya N, Tateda K, Hanson ND, Shimizu-Ibuka A. 2013. Purification, crystallization and preliminary X-ray analysis of IMP-18, a class B carbapenemase from Pseudomonas aeruginosa. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:1397–1400. doi: 10.1107/S1744309113030480. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11.Otwinowski Z, Minor W. 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276:307–326. doi: 10.1016/S0076-6879(97)76066-X. [DOI] [PubMed] [Google Scholar]

[B12] 12.Battye TG, Kontogiannis L, Johnson O, Powell HR, Leslie AG. 2011. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D Biol Crystallogr 67:271–281. doi: 10.1107/S0907444910048675. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13.Collaborative Computational Project, Number 4. 1994. The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50:760–763. doi: 10.1107/S0907444994003112. [DOI] [PubMed] [Google Scholar]

[B14] 14.Arnold K, Bordoli L, Kopp J, Schwede T. 2006. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201. doi: 10.1093/bioinformatics/bti770. [DOI] [PubMed] [Google Scholar]

[B15] 15.Yamaguchi Y, Kuroki T, Yasuzawa H, Higashi T, Jin W, Kawanami A, Yamagata Y, Arakawa Y, Goto M, Kurosaki H. 2005. Probing the role of Asp-120(81) of metallo-β-lactamase (IMP-1) by site-directed mutagenesis, kinetic studies, and X-ray crystallography. J Biol Chem 280:20824–20832. doi: 10.1074/jbc.M414314200. [DOI] [PubMed] [Google Scholar]

[B16] 16.Vagin A, Teplyakov A. 2010. Molecular replacement with MOLREP. Acta Crystallogr D Biol Crystallogr 66:22–25. doi: 10.1107/S0907444909042589. [DOI] [PubMed] [Google Scholar]

[B17] 17.Emsley P, Lohkamp B, Scott WG, Cowtan K. 2010. Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66:486–501. doi: 10.1107/S0907444910007493. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B18] 18.Murshudov GN, Vagin AA, Dodson EJ. 1997. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53:240–255. doi: 10.1107/S0907444996012255. [DOI] [PubMed] [Google Scholar]

[B19] 19.Lovell SC, Davis IW, Arendall WB III, de Bakker PI, Word JM, Prisant MG, Richardson JS, Richardson DC. 2003. Structure validation by Cα geometry: ϕ,ψ and Cβ deviation. Proteins 50:437–450. doi: 10.1002/prot.10286. [DOI] [PubMed] [Google Scholar]

[B20] 20.Concha NO, Janson CA, Rowling P, Pearson S, Cheever CA, Clarke BP, Lewis C, Galleni M, Frere JM, Payne DJ, Bateson JH, Abdel-Meguid SS. 2000. Crystal structure of the IMP-1 metallo β-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor: binding determinants of a potent, broad-spectrum inhibitor. Biochemistry 39:4288–4298. doi: 10.1021/bi992569m. [DOI] [PubMed] [Google Scholar]

[B21] 21.Palzkill T. 2013. Metallo-β-lactamase structure and function. Ann N Y Acad Sci 1277:91–104. doi: 10.1111/j.1749-6632.2012.06796.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22.Concha NO, Rasmussen BA, Bush K, Herzberg O. 1996. Crystal structure of the wide-spectrum binuclear zinc β-lactamase from Bacteroides fragilis. Structure 4:823–836. doi: 10.1016/S0969-2126(96)00089-5. [DOI] [PubMed] [Google Scholar]

[B23] 23.Scrofani SD, Chung J, Huntley JJ, Benkovic SJ, Wright PE, Dyson HJ. 1999. NMR characterization of the metallo-β-lactamase from Bacteroides fragilis and its interaction with a tight-binding inhibitor: role of an active-site loop. Biochemistry 38:14507–14514. doi: 10.1021/bi990986t. [DOI] [PubMed] [Google Scholar]

[B24] 24.Moali C, Anne C, Lamotte-Brasseur J, Groslambert S, Devreese B, Van Beeumen J, Galleni M, Frere JM. 2003. Analysis of the importance of the metallo-β-lactamase active site loop in substrate binding and catalysis. Chem Biol 10:319–329. doi: 10.1016/S1074-5521(03)00070-X. [DOI] [PubMed] [Google Scholar]

[B25] 25.Materon IC, Beharry Z, Huang W, Perez C, Palzkill T. 2004. Analysis of the context dependent sequence requirements of active site residues in the metallo-β-lactamase IMP-1. J Mol Biol 344:653–663. doi: 10.1016/j.jmb.2004.09.074. [DOI] [PubMed] [Google Scholar]

PERMALINK

Structural and Mutagenic Analysis of Metallo-β-Lactamase IMP-18

Takamitsu Furuyama

Haruka Nonomura

Yoshikazu Ishii

Nancy D Hanson

Akiko Shimizu-Ibuka

Abstract

INTRODUCTION

MATERIALS AND METHODS