Wild-type L1 was over-expressed in Escherichia coli and purified as previously described 36. This procedure produced an average of 50–60 mg of >90% pure, active protein per 4 L of growth culture. Circular dichroism spectra were collected on wild-type samples to ensure L1 expressed using the pET26b expression system had the correct secondary structure. The CD spectrum of wild type L1 showed an intense, broad feature at 190 nm and a smaller feature at 215 nm (see Additional file 1: CD spectra). These features are consistent with a sample with significant α/β content. The Compton and Johnson algorithm 38 was used to estimate secondary structure in the samples; wild-type L1 was estimated to have 38.3% α-helix, 26.7% β-structure (9.3% antiparallel β-sheet, 2.1% parallel β-sheet, and 15.3% β-turn), and 34.9% other structure. These estimates are in excellent agreement with the crystallographically determined secondary structure of ~40% α-helix and 30% β-structure 37. Metal analyses on multiple preparations of wild-type L1 demonstrated that the enzyme binds 1.9 ± 0.2 Zn(II) ions per monomer (Table 2), in agreement with previous results 36.

Steady state kinetic studies were performed on multiple preparations of wild type L1, and the resulting kinetic data are shown in Tables 3,4,5. When using nitrocefin as substrate and 50 mM cacodylate, pH 7.0, as buffer, wild-type L1 exhibited a k_cat value of 38 ± 1 s^-1 and a K_m value of 12 ± 1 μM. The inclusion of 100 μM ZnCl₂ in the assay buffer resulted in slightly lower values of K_m and higher values for k_cat36. The inclusion of higher concentrations of Zn(II) did not further affect the steady-state kinetic constants. Apparently, the purified, recombinant enzyme does not bind its full complement of Zn(II); therefore, 100 μM Zn(II) was included in all subsequent kinetic studies.

Wild-type L1 exhibited k_cat values of 41 ± 1 s^-1, 1.9 ± 0.1 s^-1, 42 ± 1 s^-1, and 82 ± 5 s^-1 for the cephalosporins, nitrocefin, cefoxitin, cefaclor, and cephalothin. For these same substrates, the K_m values were 4 ± 1 μM, 1.1 ± 0.1 μM, 13 ± 1 μM, and 8.9 ± 1.5 μM, respectively. Two penicillins were tested as substrates, and penicillin G and ampicillin exhibited K_m values of 38 ± 12 μM and 55 ± 5 μM and k_cat values of 600 ± 100 s^-1 and 520 ± 10 s^-1, respectively (Table 4). Three carbapenems were also used as substrates for L1, and biapenem, imipenem, and meropenem exhibited K_m values of 32 ± 1 μM, 57 ± 7 μM, and 15 ± 4 μM and k_cat values of 134 ± 4 s^-1, 370 ± 5 s^-1, and 157 ± 9 s^-1, respectively (Table 5). L1's preference for penicillins and carbapenems over cephalosporins, as exemplified by the k_cat values, is in agreement with previous studies and supports L1's placement in the β-lactamase 3c family 9.

Rapid-scanning visible spectra of 25 μM wild-type L1 with 5 μM nitrocefin demonstrated a decrease in absorbance at 390 nm, an increase at 485 nm, and a rapid increase and slower decrease in absorbance at 665 nm. These spectra are similar to those previously reported for wild-type L1 and nitrocefin 39, and the features can be attributed to substrate decay, product formation, and intermediate formation and decay, respectively. Under these conditions, 2.2 μM intermediate was formed during the first 10 milliseconds of the reaction (Figure 2), and the rate of decay of this intermediate corresponds to the steady-state k_cat (Table 3). To probe further the binding of nitrocefin to wild-type L1, stopped-flow fluorescence studies were conducted as previously described 40 (Figure 3). The reaction of wild-type L1 with nitrocefin under steady-state conditions at 10°C resulted in a rapid decrease in fluorescence followed by a rate-limiting return of fluorescence (Figure 3A). Fitting of the data, as described by Spencer et al.40, resulted in a K_S value for wild-type L1 of 38 ± 5 μM (Figure 3B).

(the BBL numbering scheme proposed in reference 41 was used throughout this manuscript). All sequenced subclass Ba and Bb metallo-β-lactamases (except VIM-1) have a lysine residue at position 224 41, and all computational models for substrate binding to the metallo-β-lactamases assume that the invariant carboxylate on substrates forms an electrostatic interaction with this lysine. In L1, the residue at position 224 is a serine 35, and the substrate-binding model for L1 predicts that this serine residue interacts with the carboxylate on substrate via a water molecule 37. To test the proposed role of Ser224 in L1, serine was changed to an alanine (S224A), aspartic acid (S224D), and lysine (S224K), and these mutants were characterized using metal analyses, CD spectroscopy, steady-state kinetics, and pre-steady state kinetic studies.

Small-scale growth cultures showed that all three mutants were over-expressed at levels comparable to those of wild-type L1. Large-scale over-expression and purification of the mutants showed that all three mutants were isolatable at levels comparable to those of wild-type L1. Metal analyses of the S224A and S224D mutants showed that both mutants bind nearly two Zn(II) ions (Table 2), like wild-type L1 36; however, the S224K mutant binds only 1.0 Zn(II) per protein. CD spectra of the mutants were similar to those of wild-type L1 (see Figure in Additional materials). Steady-state kinetic studies were conducted with all three mutants in buffer containing 100 μM ZnCl₂ to ensure that both Zn(II) binding sites were saturated in these studies. Addition of higher concentrations of Zn(II) did not result in different values for the steady-state kinetic constants in Tables 3,4,5.

When the cephalosporins were used as substrates, the S224A and S224K mutants exhibited 2- to 4-fold changes in K_m values (Table 3). In studies with cefoxitin, cefaclor, and cephalothin as substrate, the observed k_cat values for the S224A and S224K mutants were 2- to 7-fold lower; however, the k_cat values when using nitrocefin as substrate were slightly higher (< 2-fold). On the other hand, the S224D mutant exhibited 3- to 50-fold higher K_m values and 2- to 20-fold lower k_cat values for the cephalosporins tested. A similar trend was observed in kinetic studies when using penicillins as substrates (Table 4). Generally, the S224A and S224K mutants exhibited small changes in K_m and k_cat, while the S224D mutant yielded 20- to 40-fold increased values for K_m and >10-fold decreases in k_cat when using the penicillins as substrates. When the carbapenems were used as substrates however, the changes in K_m values were relatively smaller than with the other substrates, and 2- to 37-fold changes in k_cat were observed (Table 5).

Rapid-scanning Vis studies of the S224X mutants were conducted to probe whether the mutations caused changes in the amount of intermediate that accumulates during catalysis. When 50 μM S224A was reacted with 5 μM nitrocefin, 1.7 μM intermediate formed during the first 10 milliseconds of the reaction (Figure 2), and rate of decay of this intermediate was equal to the steady-state k_cat (Table 3). In spite of utilizing a number of reaction conditions, the S224K and S224D mutants yielded rapid-scan spectra with no detectable absorbances at 665 nm (Figure 2), indicating that the intermediate is not stabilized as well in these mutants as in wild-type L1. Stopped-flow fluorescence studies at 10°C with the S224A, S224D, and S224K mutants and nitrocefin as the substrate resulted in K_S values of 39 ± 10, 213 ± 63, and 33 ± 5 μM, respectively.

Two-thirds of all sequenced metallo-β-lactamases have an Asn at position 233 41, and this residue was predicted 42 and shown 43 to be involved with substrate binding and activation by interacting electrostatically with the substrate β-lactam carbonyl. However, in L1, Asn233 is 14 Å away from the modeled position of the substrate β-lactam carbonyl 37. To test the role of Asn233 in substrate binding, the Asn was changed to a leucine (N233L) and to an aspartic acid (N233D), and these mutants were characterized by using metal analyses, CD spectroscopy, steady-state kinetics, and pre-steady state kinetic studies.

Small-scale growth cultures showed that both mutants were over-expressed at levels comparable to that of wild-type L1. Large-scale over-expression and purification of the mutants showed that both mutants were isolatable at levels comparable to that of wild-type L1. Metal analyses of the N233L and N233D mutants showed that both bind nearly two Zn(II) ions (Table 2), like wild-type L1 36. CD spectra of the mutants were similar to those of wild-type L1. Steady-state kinetic studies were conducted with both mutants in buffer containing 100 μM ZnCl₂ to ensure that both Zn(II) binding sites were saturated in these studies. Addition of higher concentrations of Zn(II) did not result in different values for the steady-state kinetic constants in Tables 3,4,5.

With all substrates tested, the N233L and N233D mutants exhibited K_m values that differed less than a factor of 4 than that observed for wild-type L1 (Tables 3,4,5). The k_cat values exhibited by these mutants for all substrates also differed by less than a factor of 4, except when biapenem and meropenem were used as substrates for the N233D mutant. With these two substrates, there was a 19-fold and 45-fold decrease in the k_cat values when using biapenem and meropenem, respectively (Table 5). The steady-state kinetic data generally support the prediction that Asn233 does not play a large role in binding or catalysis.

However, rapid-scanning Vis studies of N233L and N233D with nitrocefin demonstrate that no detectable amounts of intermediate are formed during the reaction, even when using a wide number of reaction conditions (Figure 2). Stopped-flow fluorescence studies at 10°C with the N233L and N233D mutants and nitrocefin as substrate resulted in K_S values of 26 ± 9 and 25 ± 8 μM, respectively.

The substrate-binding model showed that Tyr228 in L1 was position-conserved with Asn233 in the other crystallographically characterized metallo-β-lactamases 3742444546. Spencer and coworkers postulated that Tyr228 is part of an oxyanion hole that interacts with the β-lactam carbonyl on substrate and helps to stabilize the putative tetrahedral intermediate formed during substrate turnover 37. To test this hypothesis, Tyr228 was changed to an alanine and to a phenylalanine to afford the Y228A and Y228F mutants, respectively.

Small-scale growth cultures showed that both mutants were over-expressed at levels comparable to those of wild-type L1. Large-scale over-expression and purification of the Y228A and Y228F mutants showed that both mutants were isolatable at levels comparable to those of wild-type L1. Metal analyses of the mutants showed that both bind nearly two Zn(II) ions (Table 2), like wild-type L1 36, and CD spectra of the mutants were similar to those of wild-type L1. Steady-state kinetic studies were conducted with both mutants in buffer containing 100 μM ZnCl₂ to ensure that both Zn(II) binding sites were saturated in these studies. Addition of higher concentrations of Zn(II) did not result in different values for the steady-state kinetic constants in Tables 3,4,5.

When cephalosporins were used as substrates, the Y228A and Y228F mutants exhibited K_m values that were 6- to 45-fold higher than those observed for wild-type L1 (Table 3). The largest change in K_m was observed when cefaclor was used as substrate, and the smallest change was observed when nitrocefin was used as substrate. The Tyr228 mutants exhibited < 4-fold change in k_cat values for the cephalosporins tested (Table 3), suggesting that Tyr228 is not playing a large role in catalysis. When penicillins were used as substrates, the Tyr228 mutants exhibited 3- to 13-fold increased K_m values and < 2-fold changes in k_cat, as compared to the values ascertained using wild-type L1 (Table 4). On the other hand when carbapenems were used as substrates, the Tyr228 mutants exhibited < 6-fold increases in K_m values as compared to those values for wild-type L1 (Table 5). Interestingly, there was a 2- to 8-fold drop in k_cat values for the Tyr228 mutants, as compared to values observed for wild-type L1, when using the carbapenems as substrates.

Rapid-scanning Vis spectra of the reaction of the Y228A and Y228F mutants with nitrocefin demonstrated a marked decrease in the amount of intermediate formed with these mutants (Figure 2). In reactions with 50 μM mutant and 5 μM nitrocefin, only 0.75 and < 0.30 μM intermediate formed for the Y228F and Y228A mutants, respectively. The concentration of mutants were varied between 25 to 150 μM to ensure that all of the substrate was bound; however, none of the reactions resulted in the detection of intermediate at levels observed for wild-type L1 (data not shown). Stopped-flow fluorescence studies at 10°C of nitrocefin hydrolysis by Y191A and Y191F resulted in K_S values of 85 ± 9 and 22 ± 6 μM, respectively.

All crystallographically characterized metallo-β-lactamases have a flexible amino acid chain that extends over the active site 3742444546474849. Previous NMR studies on CcrA have shown that this loop "clamps down" on substrate or inhibitor upon binding, and there is speculation that the distortion of substrate upon clamping down of the loop may drive catalysis 50. The crystal structure of L1 showed that there is a large loop that extends over the active site, and modeling studies have predicted that two residues, Ile164 and Phe158, make significant contacts with large, hydrophobic substituents at the 2' or 6' positions on penicillins, cephalosporins, or carbapenems 37. To test this prediction, Ile 164 and Phe158 were changed from large, hydrophobic residues to alanines to afford the I164A and F158A mutants.

Small-scale growth cultures demonstrated the I164A and F158A mutants were over-expressed at levels comparable to that of wild-type L1 (data not shown). Large-scale over-expression and purification of the mutants resulted in comparable quantities of isolatable enzymes, which had identical CD spectra as wild-type L1 and bound slightly less Zn(II) than wild-type L1 (Table 2). All steady-state kinetic studies were conducted in buffers containing 100 μM ZnCl₂ to ensure that both metal binding sites were saturated during the studies.

When using the cephalosporins, nitrocefin, cefoxitin, and cephalothin, as substrates and the I164A mutant, there were 2- to 10-fold (only for cefoxitin) increases in K_m and 2- to 4-fold increases in k_cat observed (Table 3). However when cefaclor was used as substrate, the I164A mutant exhibited a 3-fold decrease in K_m and a 1.5-fold decrease in k_cat (Table 3). On the other hand, the K_m and k_cat values for the I164A mutant when the penicillins or carbapenems were used as substrates were very similar to those numbers exhibited by wild-type L1 (Tables 4 and 5).

When the cephalosporins were used as substrates for the F158A mutant, the K_m values observed were 7- to 31-fold higher than those determined for wild-type L1, and surprisingly, the k_cat values were 2- to 31-fold higher than those exhibited by wild-type L1 (Table 3). As with the I164A mutant, the changes in K_m and k_cat for the penicillins and carbapenems were relatively small, as compared with the values obtained with the cephalosporins (Table 4).

Rapid-scanning Vis studies on nitrocefin hydrolysis by I164A and F158A showed a marked decrease in intermediate accumulation, with the I164A mutant generating < 0.30 μM intermediate and the F158A producing no detectable intermediate (Figure 2). Stopped-flow fluorescence studies at 10°C resulted in a K_S value of 31 ± 11 μM for the I164A mutant. The reaction of F158A with nitrocefin was so rapid, we could not determine a K_S value for this mutant.

E. coli strains DH5α and BL21(DE3) pLysS were obtained from Gibco BRL and Novagen, respectively. Plasmids pET26b and pUC19 were purchased from Novagen. Primers for sequencing and mutagenesis studies were purchased from Integrated DNA Technologies. Deoxynucleotide triphosphates (dNTP's), MgSO₄, thermopol buffer, Deep Vent DNA polymerase, and restrictions enzymes were purchased from Promega or New England Biolabs. Polymerase chain reaction was conducted using a Thermolyne Amplitron II unit. DNA was purified using the Qiagen QIAQuick gel extraction kit or Plasmid Purification kit with QIAGEN-tip 100 (Midi) columns. Wizard Plus Minipreps were acquired from Promega. Luria-Bertani (LB) media was made following published procedures 67. Isopropyl-β-thiogalactoside (IPTG), Biotech grade, was procured from Anatrace. Phenylmethylsulfonylfluoride (PMSF) was purchased from Sigma. Protein solutions were concentrated with an Amicon ultrafiltration cell equipped with YM-10 DIAFLO membranes from Amicon, Inc. Dialysis tubing was prepared using Spectra/Por regenerated cellulose molecular porous membranes with a molecular weight cut-off of 6–8,000 g/mol. Q-Sepharose Fast Flow was purchased from Amersham Pharmacia Biotech. Nitrocefin was purchased from Becton Dickinson, and solutions of nitrocefin were filtered through a Fisherbrand 0.45 micron syringe filter. Cefaclor, cefoxitin, and cephalothin were purchased from Sigma; penicillin G and ampicillin were purchased from Fisher. Imipenem, meropenem, and biapenem were generously supplied by Merck, Zeneca Pharmaceuticals, and Lederle (Japan), respectively. All buffers and media were prepared using Barnstead NANOpure ultrapure water.

The over-expression plasmid for L1, pUB5832, was digested with NdeI and HindIII, and the resulting ca. 900 bp piece was gel purified and ligated using T4 ligase into pUC19, which was also digested with NdeI and HindIII, to yield the cloning plasmid pL1PUC19. Mutations were introduced into the L1 gene by using the overlap extension method of Ho et al.60, as described previously 68. The oligonucleotides used for the preparation of the mutants are shown in Table 1. The ca. 900 bp PCR products were digested with NdeI and HindIII and ligated into pUC19. The DNA sequences were analyzed by the Biosynthesis and Sequencing Facility in the Department of Biological Chemistry at Johns Hopkins University. After confirmation of the sequence, the mutated pL1PUC19 plasmid was digested with NdeI and HindIII, and the 900 bp, mutated L1 gene was gel purified and ligated into pET26b to create the mutant overexpression plasmids. To test for overexpression of the mutant enzymes, E. coli BL21(DE3)pLysS cells were transformed with the mutated over-expression plasmids, and small scale growth cultures were used 68. Large-scale (4 L) preparations of the L1 mutants were performed as described previously 36. Protein purity was ascertained by SDS-PAGE.

The concentrations of L1 and the mutants were determined by measuring the proteins' absorbance at 280 nm and using the published extinction coefficient of ε₂₈₀ nm = 54,804 M^-1•cm^-136 or by using the method of Pace 69. Before metal analyses, the protein samples were dialyzed versus 3 × 1 L of metal-free, 50 mM HEPES, pH 7.5 over 96 hours at 4°C. A Varian Inductively Coupled Plasma Spectrometer with atomic emission spectroscopy detection (ICP-AES) was used to determine metal content of multiple preparations of wild type L1 and L1 mutants. Calibration curves were based on three standards and had correlation coefficient limits of at least 0.9950. The final dialysis buffer was used as a blank, and the Zn(II) content in the final dialysis buffers was shown to be < 0.5 μM (detection limit of ICP) in separate ICP measurements. The emission line of 213.856 nm is the most intense for zinc and was used to determine the Zn content in the samples. The errors in metal content data reflect the standard deviation (σ_n-1) of multiple enzyme preparations.

Steady-state kinetic assays were conducted at 25°C in 50 mM cacodylate buffer, pH 7.0, containing 100 μM ZnCl₂ on a HP 5480A diode array UV-Vis spectrophotometer at 25°C. The changes in molar absorptivities (Δε) used to quantitate products were (in M^-1cm^-1): nitrocefin, Δε₄₈₅ = 17,420; cephalothin, Δε₂₆₅= -8,790; cefoxitin, Δε₂₆₅= -7,000; cefaclor, Δε₂₈₀= -6,410; imipenem, Δε₃₀₀= -9,000; meropenem, Δε₂₉₃= -7,600; biapenem, Δε₂₉₃= -8,630; ampicillin, Δε₂₃₅= -809; and penicillin G, Δε₂₃₅ = -936. When possible, substrate concentrations were varied between 0.1 to 10 times the K_m value. In kinetic studies using substrates with low K_m values (cefoxitin, nitrocefin, and cephalothin) or with small Δε values (penicillin and ampicillin), we typically used substrate concentrations varied between ~ K_m and 10 × K_m and used as much of the ΔA versus time data (that was linear) as possible to determine the velocity. Steady-state kinetics constants, K_m and k_cat, were determined by fitting initial velocity versus substrate concentration data directly to the Michaelis equation using CurveFit 36. The reported errors reflect fitting uncertainties. All steady-state kinetic studies were performed in triplicate with recombinant L1 from at least three different enzyme preparations.

Circular dichroism samples were prepared by dialyzing the purified enzyme samples versus 3 × 2 L of 5 mM phosphate buffer, pH 7.0 over six hours. The samples were diluted with final dialysis buffer to ~75 μg/mL. A JASCO J-810 CD spectropolarimeter operating at 25°C was used to collect CD spectra.

Rapid-scanning Vis spectra of nitrocefin hydrolysis by L1 and the L1 mutants were collected on a Applied Photophysics SX.18MV stopped-flow spectrophotometer equipped with an Applied Photophysics PD.l photodiode array detector and a 1 cm pathlength optical cell. A typical experiment consisted of 25 μM enzyme and 5 μM nitrocefin in 50 mM cacodylate buffer, pH 7.0 containing 100 μM ZnCl₂, the reaction temperature was thermostated at 25°C, and the spectra were collected between 300 and 725 nm. Data from at least three experiments were collected and averaged. Absorbance data were converted to concentration data as described previously by McMannus and Crowder 39. Stopped-flow fluorescence studies of nitrocefin hydrolysis by L1 were performed on an Applied Photophysics SX.18MV spectrophotometer, using an excitation wavelength of 295 nm and a WG320 nm cut-off filter on the photomultiplier. These experiments were conducted at 10°C using the same buffer in the rapid-scanning Vis studies. Fluorescence data were fitted to k_obs = {(k_f [S]) / K_S + [S])} + k_r as described previously 40 or to k_obs = k_f [S] + k_r by using CurveFit v. 1.0.