Analysis of the Functional Contributions of Asn233 in Metallo-β-Lactamase IMP-1

Abstract

Metallo-β-lactamases, such as IMP-1, are a major global health threat, as they provide for bacterial resistance to a wide range of β-lactam antibiotics, including carbapenems. Understanding the molecular details of the enzymatic process and the sequence requirements for function are essential aids in overcoming β-lactamase-mediated resistance. An asparagine residue is conserved at position 233 in approximately 67% of all metallo-β-lactamases. Despite its conservation, the molecular basis of Asn233 function is poorly understood and remains controversial. It has previously been shown that mutations at this site exhibit context-dependent sequence requirements in that the importance of a given amino acid depends on the antibiotic being tested. To provide a more thorough examination as to the function and sequence requirements at this position, a collection of IMP-1 mutants encoding each of the 19 possible amino acid substitutions was generated. The resistance levels toward four β-lactam antibiotics were measured for Escherichia coli containing each of these mutants. The sequence requirements at position 233 for wild-type levels of resistance toward two cephalosporins were the most relaxed, while there were more stringent sequence requirements for resistance to ampicillin or imipenem. Enzyme kinetic analysis and determinations of steady-state protein levels indicated that the effects of the substitutions on resistance are due to changes in the kinetic parameters of the enzyme. Taken together, the results indicate that substitutions at position 233 significantly alter the kinetic parameters of the enzyme, but most substituted enzymes are able to provide for a high level of resistance to a broad range of β-lactams.

INTRODUCTION

The β-lactams, which include the penicillins and cephalosporins, are the most widely used class of antibiotics, and resistance to these drugs is a significant clinical problem (7, 25). β-Lactamases are secreted into the periplasm and hydrolyze the conserved four-membered β-lactam ring, rendering the drug inactive. Currently, there are hundreds of β-lactamases reported, and they are regarded as the most common mechanism of resistance to this class of antibiotics (43).

There are four classes of β-lactamases (A to D) based on primary amino acid sequence homology. β-Lactamases in classes A, C, and D hydrolyze the drugs by using a catalytic serine as the primary nucleophile (9, 15). Class B β-lactamases are metallo-enzymes that require either one or two zinc ions in the active site. The zinc ions in the active site decrease the pK of a coordinated water molecule, resulting in a hydroxide ion that acts as a nucleophile to attack the carbonyl group of the β-lactams (14). These enzymes exhibit a broad substrate profile, including carbapenems, which are often held in reserve as antibiotics of last resort (18, 26). Mechanism-based inhibitors of the serine-active β-lactamases are available for clinical use, but these molecules do not act on class B enzymes (2). The genes encoding metallo-β-lactamases are often found on mobile genetic units, such as transposons and plasmids. For example, the bla_IMP-1 gene was the first transferable metallo-β-lactamase identified and was found within an integron in clinical isolates of Serratia marcescens (22, 33). The widespread use of β-lactams creates the selective pressure to drive the dissemination of these genes to bacterial pathogens, such as Enterobacteriaceae (7).

The class B enzymes are included in the metallo-β-lactamase superfamily of proteins (6,000 members) that share a similar αββα scaffold and are largely binuclear enzymes with a diverse range of catalytic functions, e.g., endoribonuclease, thiol-ester hydrolase, and oxidoreductase activities (2, 31). Metallo-β-lactamases are further divided into three subclasses, B1, B2 and B3, based on three distinct lineages determined by their primary sequence homologies (16). Most known metallo-β-lactamases are from subclass B1, including the IMP, VIM, and emerging NDM-1 enzymes (24, 33, 47). IMP-1 is considered a model subclass B1 metallo-β-lactamase and has been the focus of several structural and site-directed mutagenesis studies (13, 19, 20, 27, 28, 32). The exact mechanisms and functional significance of several residues found in the active site, however, remain either unclear or controversial (11, 13, 20, 32).

Asn233 (class B β-lactamase numbering scheme [16]) has been shown to be an interesting residue among metallo-β-lactamases. Asn233 is found on the L3 loop of metallo-β-lactamases, which has been shown to affect the substrate specificity of the enzyme (16, 29) (Fig. 1). It is conserved in approximately 67% of all class B β-lactamases and is found in all but one IMP variant (IMP-14) (8, 39). This conservation is striking in that overall sequence identity among all class B enzymes is approximately 11%. Even within subclass B1 enzymes, overall sequence identity is only about 23%. This suggests that Asn233 plays a significant role in the function of the enzyme (2).

Fig. 1.

Diagram of the IMP-1 metallo-β-lactamase structure. The two zinc ions are represented as gray spheres in the active-site pocket. The asparagine at position 233 is labeled adjacent to the zinc ions. The coordinates for the IMP-1 wild-type structure were obtained from the Protein Data Bank (1DDK).

Several mechanisms for class B β-lactamases have been proposed and differ between subclasses (for a review, see reference 2). While there have been attempts to elucidate these mechanisms, there is little agreement on the function of the conserved asparagine at position 233 (13, 20, 27, 29). For example, Asn233 has been proposed to be involved in either the substrate binding or catalytic mechanism (13, 32, 34, 36, 44, 46). In contrast, it has been suggested that Asn233 has no direct function (11, 13, 17, 40, 45). The hypothesis that Asn233 does not have a role in substrate binding and catalysis, however, does not explain the sequence conservation at the position.

To provide a detailed and comprehensive examination of the 19 substitutions possible at this position in the model subclass B1 IMP-1 β-lactamase, a collection of mutants was created to encode all of the possible amino acid substitutions at position 233. The mutants were tested for their abilities to confer resistance for Escherichia coli. The resistance levels were then compared to the in vivo steady-state protein expression levels and kinetic parameters of several IMP-1 β-lactamase variants. The results of these experiments indicated that, in general, substitutions at position 233 did not alter protein expression levels but did impact enzyme kinetic parameters for various substrates.

MATERIALS AND METHODS

Site-directed mutagenesis.

The collection of IMP-1 position 233 mutants were constructed by overlap-extension PCR using Klentaq DNA polymerase (21). Nineteen different oligonucleotide primer pairs were used to replace the wild-type asparagine codon with that for the alternative amino acids. The PCR product was ligated into the pTP123-IMP-1 plasmid and electroporated into E. coli XL1-Blue cells (Stratagene) (38). The colonies were selected on LB agar supplemented with 12.5 μg/ml chloramphenicol (6). DNA sequencing was used to confirm the sequence of the gene and the presence of the desired mutation.

Immunoblot analysis.

The effect of amino acid substitutions at position 233 on steady-state expression levels of IMP-1 β-lactamase was determined by immunoblot analysis of whole-cell lysates and periplasmic fractions of E. coli XL1-Blue expressing each of the IMP-1 variants. Cultures were grown to log phase in LB agar supplemented with chloramphenicol and 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside). Whole-cell lysates were prepared by resuspending the cell pellets (adjusted for cell density to ensure equal loading) in Laemmli buffer. The periplasmic contents were released by osmotic shock, and the total protein content was determined by Bradford assay. The loading volumes were adjusted to ensure equal protein contents (3, 30). The whole-cell lysates and periplasmic preparations were resolved on an SDS-PAGE gel and electrotransferred to a nitrocellulose membrane (Amersham; GE Healthcare, Piscataway, NJ). The membrane was probed with a rabbit monoclonal anti-StrepII horseradish peroxidase (HRP)-conjugated antibody that recognizes the Strep-tag present on the IMP-1 enzyme (Novagen).

MIC determinations.

The MIC values were determined as previously described (27). In summary, 1 × 10⁴ E. coli XL1-Blue cells/ml harboring the plasmids encoding either wild-type or a mutant IMP-1 gene were inoculated into LB broth supplemented with 2-fold dilutions of either cefotaxime, cephalothin, or ampicillin. Etest strips (bioMérieux) were used to determine the MIC values for imipenem toward the IMP-1 variants in accordance with the manufacturer's protocol, with the exception that LB agar was used in place of Muller-Hinton agar.

Purification of IMP-1 wild-type and mutant β-lactamases.

Plasmids encoding either the wild-type or mutant variants of IMP-1 were transformed into E. coli RB791 (strain W3110 lacI^qL8) (1, 4). Protein expression was induced at mid-log phase by the addition of 1 mM IPTG. The cells were harvested by centrifugation after 14 to 18 h, and the periplasmic contents were released by osmotic shock (30). The IMP-1 wild-type and mutant β-lactamases were purified using a C-terminal StrepII-tag that allowed for the purification of the β-lactamases to >90% homogeneity in one step using Strep-tactin Superflow agarose (Novagen). These steps were performed in accordance with the manufacturer's protocol, except that EDTA was not added to the buffers.

Kinetic parameters.

Enzyme kinetic parameters were determined as previously described (27). In summary, hydrolysis of ampicillin (Δε_{235 nm} = −900 M⁻¹cm⁻¹), imipenem (Δε_{300 nm} = −9,000 M⁻¹cm⁻¹), cephalothin (Δε_{262 nm} = −7,660 M⁻¹cm⁻¹), and cefotaxime (Δε_{264 nm} = −7,250 M⁻¹cm⁻¹) by wild-type and substituted IMP-1 enzymes was measured using a DU800 spectrophotometer. The reactions were performed in 50 mM HEPES (pH 7.5), 50 μM ZnSO₄, and 20 μg/ml bovine serum albumin (BSA) at 30°C. The initial velocities were measured and fitted to the Michaelis-Menton equation v = k_cat [S]/(K_m +[S]), using GraphPad Prism5 to determine k_cat and K_m. When V_max could not be determined, the catalytic efficiency (k_cat/K_m) was estimated using the equation v = k_cat/K_m [E][S], where [S] ≪ K_m. The initial velocities were measured in at least duplicate and averaged to determine the kinetic parameters.

RESULTS AND DISCUSSION

Susceptibility testing of IMP-1 Asn233 variants.

It has been postulated that Asn233 plays an important role in metallo-β-lactamase catalysis, although there is not a consensus as to its exact function (11, 13, 27, 46). Some possible functions include a role in substrate recognition or a contribution to the oxyanion hole. Alternatively, Asn233 may not play a role in catalysis. Previously, we randomized the codon for position 233 to create a library containing all possible amino acid substitutions and identified functional mutants by introducing the library into E. coli and selecting for clones that were resistant to representative β-lactam antibiotics from the penicillin, cephalosporin, and carbapenem classes (27). The bla_IMP-1 gene from functional mutants was sequenced, and it was found that position 233 displayed context-dependent sequence requirements in that the identity of the substitutions discovered at a position varied depending on the antibiotic used in the selection. However, a systematic assessment of the sequence requirements at position 233 for hydrolysis of the various classes of β-lactam antibiotics has not been performed.

To address this issue, the effect of each possible substitution at position 233 on IMP-1-mediated β-lactam antibiotic resistance was examined. Oligonucleotides encoding each of the 19 non-wild-type amino acids at position 233 were synthesized and used with overlap extension PCR to create the set of mutants (21). E. coli cells harboring the bla_IMP-1 wild-type and each of the 19 variants were examined individually for resistance levels toward ampicillin, imipenem, cefotaxime, and cephalothin, which represent the three major classes of β-lactam antibiotics, including two cephalosporins (Fig. 2). The MIC results are summarized in Table 1.

Fig. 2.

Structures of the β-lactam antibiotic substrates used in this study.

Table 1.

MICs for E. coli containing IMP-1 β-lactamase and the amino acid variants at position 233

Plasmid	MIC (μg/ml)a
	AMP	IMP	CEF	CTX
pTP123	<2.0	0.25	16	<2.0
IMP-1	125	3.0	500	250
N233A	31	0.25	125	63
N233C	<2.0	0.25	31	<2.0
N233D	250	3.0	1,000	500
N233E	125	3.0	500	250
N233F	125	3.0	500	250
N233G	125	0.38	1,000	500
N233H	125	0.50	1,000	125
N233I	16	0.38	500	125
N233K	16	0.38	500	63
N233L	16	0.50	500	250
N233M	16	0.75	1,000	500
N233P	125	0.38	500	125
N233Q	125	2.0	1,000	500
N233R	31	0.38	500	250
N233S	125	1.5	1,000	500
N233T	16	0.38	1,000	500
N233V	31	0.38	500	500
N233W	250	1.5	500	63
N233Y	125	3.0	500	125

^aThe IMP-1 wild type and position 233 variants are encoded on plasmid pTP123 and expressed in E. coli XL1-Blue cells (6). AMP, ampicillin; IMP, imipenem; CEF, cephalothin; CTX, cefotaxime.

Consistent with our previous findings, the number of substitutions that provide β-lactam resistance at levels near wild-type levels is dependent on the antibiotic being examined. None of the 19 variants at position 233 provide resistance levels greater than 2-fold above wild-type levels regardless of the antibiotic tested. A total of 10 of the 19 IMP-1 variants, however, provide ampicillin MIC values within a 2-fold range of wild-type resistance levels (125 to 250 μg/ml). The amino acid substitutions of these 10 mutants exhibit a wide spectrum of side chain characteristics, including Asp, Gly, Pro, and Trp, among others (Table 1). Similarly, the 9 mutants that result in significantly reduced ampicillin resistance levels vary in physicochemical properties, including Ala, Cys, Met, Thr, and Val mutants. In addition, large, branched hydrophobic amino acids such as leucine and isoleucine, as well as positively charged residues, including arginine and lysine, also provide lower levels of resistance. Asn233Cys is particularly interesting, as it displays ampicillin resistance at background levels.

The hydrolysis of imipenem imposes the most stringent sequence requirements on the enzyme. Only 7 of the 19 substitutions at position 233 result in imipenem resistance levels within 2-fold of wild-type levels (1.5 to 3.0 μg/ml). These residues include Asp, Glu, Phe, Gln, Ser, Trp, and Tyr, which encompass a range of volumetric and molecular characteristics with the notable exclusion of positively charged amino acids. The imipenem MIC values of the Asn233His, Asn233Lys, and Asn233Arg mutants (0.38 to 0.50 μg/ml) are just above the background resistance levels of E. coli without a β-lactamase (0.25 μg/ml). There is significant overlap in the sequence requirements for ampicillin and imipenem resistance in that the 7 amino acids that are consistent with imipenem resistance are a subset of the 10 amino acids that are consistent with ampicillin resistance (Fig. 3).

Fig. 3.

Graphical representation of the MICs for E. coli containing the IMP-1 variants. The normalized MIC values for ampicillin, imipenem, cefotaxime, and cephalothin are shown in red, green, blue, and yellow, respectively.

MIC values for the cephalosporins, cephalothin and cefotaxime, were also obtained for E. coli cells harboring plasmids containing the wild type and each of the 19 IMP-1 variants. The sequence requirements for hydrolysis of the cephalosporins are less stringent than those for ampicillin or imipenem. E. coli containing wild-type IMP-1 provided resistance toward cephalothin with a MIC of 500 μg/ml. The majority of the substitutions (17 of 19) at position 233 result in MICs similar to that of the wild type (250 to 1,000 μg/ml), and 7 of the 19 variants exhibited a slightly increased cephalothin resistance of 1,000 μg/ml. The levels of resistance of these IMP-1 variants toward cefotaxime were similar, as 11 of the 19 variants exhibited levels similar to or higher than wild-type values. The MIC for cefotaxime against E. coli containing wild-type IMP-1 was determined to be 250 μg/ml, and the resistance levels of 15 variants were similar to that of the wild type, with cefotaxime MIC values ranging from 125 to 500 μg/ml. Seven residues, with a range of side chain properties, exhibited a 2-fold increase in cefotaxime MIC to 500 μg/ml. The Asn233Cys variant, with a cefotaxime MIC below 2.0 μg/ml, exhibited the largest reduction in β-lactamase function (Table 1 and Fig. 3).

These results demonstrate that the sequence requirements at position 233 to maintain near wild-type resistance levels vary significantly with the β-lactam antibiotic substrate. For example, the Asn233Arg IMP-1 mutant exhibited a reduced level of resistance toward ampicillin and imipenem but displayed resistance levels similar to wild-type levels for the cephalosporins examined here. In contrast, the Asn233Cys mutation resulted in a decrease in function for all of the substrates tested, suggesting that the presence of the Cys side chain at position 233 is detrimental for hydrolysis of all classes of β-lactam antibiotics.

Steady-state in vivo β-lactamase expression levels.

Single amino acid substitutions are capable of destabilizing β-lactamases and thereby drastically lowering protein levels in vivo, leading to decreased resistance (5, 41). The steady-state in vivo expression levels of IMP-1 and the 19 mutant enzymes were evaluated using whole-cell lysates and periplasmic extracts of E. coli encoding wild-type IMP-1 or the various mutants (Fig. 4). Immunoblot analysis was performed on E. coli cells carrying plasmids encoding wild-type IMP-1 and the 19 variants at position 233. As a negative control, the whole-cell and periplasmic fractions of E. coli harboring the parent pTP123 vector lacking a β-lactamase gene were included. The periplasmic contents of mid-log-phase cultures were released by osmotic shock, separated by SDS-PAGE, and probed with an anti-StrepII-tag monoclonal antibody following a transfer to a nitrocellulose membrane. This antibody detects the C-terminal StrepII-tag that is present on the wild type and all of the IMP-1 variants used in this study (see Materials and Methods).

Fig. 4.

(A) Steady-state protein levels of IMP-1 variants as analyzed by immunoblot analysis of whole-cell lysates (W) and the periplasmic contents (P) of E. coli cultures containing the pTP123 plasmid encoding the wild-type or mutant enzymes. The E. coli culture containing the empty vector pTP123 without a bla_IMP-1 insert is labeled with a minus symbol, and the pTP123 plasmid containing wild-type IMP-1 is shown labeled with a plus symbol. The lanes representing other position 233 substitutions are labeled with their corresponding single-letter amino acid code. (B) Densitometry of protein levels from the periplasmic contents in panel A, quantified using the ImageJ software program.

The immunoblotting experiments revealed for both the whole-cell lysates and the soluble, periplasmic fractions that most of the IMP-1 variants were expressed at levels similar to that of the wild type, suggesting that few mutations drastically impact the stability or solubility of the enzyme (Fig. 4). The Asn233Cys and Asn233Ile mutants are notable exceptions. While other mutants are expressed equally well in the whole-cell and periplasmic fractions, Asn233Cys and Asn233Ile levels are lower than wild-type levels in the periplasm. All other mutants exhibited similar levels compared to wild-type levels in the periplasm. The lack of effect on IMP-1 stability may be due to the fact that the Asn233 residue is surface exposed and located at the glycine-rich apex of the unstructured active-site loop (13). Consistent with these results, previous studies using circular dichroism on a limited number of substitutions at this position did not reveal a change in the overall structure of the enzyme (11).

Kinetic parameters of wild-type and IMP-1-substituted β-lactamases.

The amino acid substitutions at position 233 in IMP-1 that impair in vivo function could exert their effect through changes in stability or catalytic efficiency or both. The immunoblotting experiments suggest that the majority of substitutions do not alter levels of soluble protein expression. In order to evaluate changes in catalysis, wild-type IMP-1 and the Asn233Lys-, Asn233Arg-, Asn233Cys-, Asn233Ala-, and Asn233Glu-substituted enzymes, which exhibit a range of side chain volumes and charges, were purified, and steady-state kinetic parameters for ampicillin, imipenem, cephalothin, and cefotaxime hydrolysis were determined for each (Table 2).

Table 2.

Kinetic parameters and MICs for wild-type and mutant IMP-1 β-lactamases

β-Lactamase	Parameter	Value
		AMP	IMP	CEF	CTX
Wild type	kcat (s−1)	62 ± 2.3	98 ± 6.7	59 ± 3.8	16 ± 0.58
	Km (μM)	97 ± 16	59 ± 11	13 ± 3.7	0.98 ± 0.17
	kcat/Km (s−1μM)	0.64	1.7	4.6	17
	MIC (μg/ml)	125	3.0	500	250
N233K mutant	kcat (s−1)	132 ± 6.7	>336a	821 ± 51	595 ± 29
	Km (μM)	1,445 ± 207	>2,000a	411 ± 55	98 ± 17
	kcat/Km (s−1μM)	0.091	0.17 ± 0.015b	2.0	6.1
	MIC (μg/ml)	16	0.38	500	63
N233R mutant	kcat (s−1)	83 ± 3.7	>134a	>125a	391 ± 23
	Km (μM)	501 ± 77	>2,000a	>800a	73 ± 16
	kcat/Km (s−1μM)	0.17	0.067 ± 0.020b	0.16 ± 0.073b	5.3
	MIC (μg/ml)	31	0.38	500	250
N233C mutant	kcat (s−1)	9.1 ± 0.66	>5.4a	>64a	51 ± 6.6
	Km (μM)	1,098 ± 253	>2,000a	>800a	76 ± 20
	kcat/Km (s−1μM)	0.0083	0.0027 ± 0.00062b	0.080 ± 0.0090b	0.67
	MIC (μg/ml)	<2.0	0.25	31	<2.0
N233A mutant	kcat (s−1)	230 ± 20c	>970a,c	254 ± 34	495 ± 40c
	Km (μM)	110 ± 18c	>1,000a,c	89 ± 36	30 ± 4.0c
	kcat/Km (s−1μM)	2.1c	0.97 ± 0.30b,c	2.9	16c
	MIC (μg/ml)	31	0.25	31	<2.0
N233E mutant	kcat (s−1)	416 ± 36c	111 ± 8.0c	67 ± 3.0	34 ± 8.0c
	Km (μM)	92 ± 3.0c	178 ± 13c	16 ± 3.4	8.0 ± 2.0c
	kcat/Km (s−1μM)	4.5c	0.62c	4.1	4.3c
	MIC (μg/ml)	125	3.0	500	250

^aThe K_m value was too high to determine these values accurately.

^bThe k_cat/K_m value was determined using the equation v = k_cat/K_m [E][S] (see Materials and Methods).

^cData from Materon et al. (27).

According to the MIC results, the Asn233Lys- and Asn233Arg-substituted enzymes should exhibit very similar activities toward these antibiotics due to common, positively charged side chains. This is in fact what was observed for ampicillin, imipenem, and cefotaxime but not for cephalothin, which was hydrolyzed with 10-fold-higher catalytic efficiency by the Asn233Lys enzyme than by the Asn233Arg enzyme. Overall, the kinetic parameters of the Asn233Arg enzyme are very similar to the Asn233Lys enzyme for ampicillin, imipenem, and cefotaxime, with increases in both k_cat and K_m in comparison to wild-type IMP-1.

The cysteine substitution significantly decreased the MICs for all β-lactams tested. The Asn233Cys-substituted enzyme displayed increased K_m values for all substrates tested. The effects on k_cat, however, were substrate specific, as it was markedly decreased for ampicillin but increased for cefotaxime (Table 2). This resulted in a decrease in catalytic efficiency for all β-lactams tested, which agrees with the lower MIC values observed. Although the asparagine side chain is not close enough for a direct interaction with the zinc ions of the active site, the thiol group of the cysteine side chain could alter zinc coordination or allow aberrant substrate contacts, thereby decreasing enzymatic function.

A common trend of increased k_cat values against each β-lactam tested was observed for all enzymes except the Asn233Cys mutant. Additionally, all substitutions exhibited either no change or an increase in K_m for each substrate assayed compared to wild-type values. These results are similar to those of our previous study of the Asn233Ala and Asn233Glu mutants, which increased the k_cat and K_m values for the majority of substrates tested (27). An exception was seen for imipenem hydrolysis, where the larger increases in K_m led to decreased efficiency for both the Asn233Ala and Asn233Glu mutants. The k_cat values of the Asn233Glu-substituted enzyme for imipenem and cephalothin are similar to that of the wild type; however, the k_cat values are increased for ampicillin and cefotaxime in comparison to that of the wild type. The common trend in the kinetic parameter changes for the Asn233 mutants occurs despite the broad spectrum of positively charged, negatively charged, and nonpolar side chains represented by the mutations. Interestingly, this trend of increased k_cat and K_m was also observed for penicillin as cephalosporin substrates in the subclass B1 CcrA enzyme for the Ala, Asp, and Leu substitutions of the corresponding Asn residue (46). Yanchak et al. also demonstrated that substitutions at position 233 did not affect the rate of product formation but did affect the rate of substrate disappearance for nitrocefin hydrolysis (46). This suggests that Asn233 functions earlier in the catalytic mechanism, i.e., substrate binding or intermediate formation.

Data from other crystallographic, kinetic, and computational studies of the subclasses B1, B2, and B3 have led to suggestions that the Asn233 backbone functions in substrate binding and catalysis or, alternatively, has no direct function (11, 13, 17, 40, 45). However, the sequence conservation of an asparagine at position 233 (Fig. 5) and the substantial changes to the kinetic parameters of the substituted enzymes do not agree with this hypothesis.

Fig. 5.

Conservation of Asn233 in subclass B1 metallo-β-lactamases. A sequence alignment is shown of the loop containing position 233 in the IMP-1, IMP-14, NDM-1, BcII, VIM-1, CcrA, and BlaB metallo-enzymes using the GenBank accession numbers AY168635, AY553332.1, HQ738352.1, M11189, Y18050, M63556, and AF189298, respectively. The alignment was performed using the ClustalW2 program (12).

Molecular dynamics of the IMP-1 enzyme suggest that both the side chain and main chain of Asn233 are involved in substrate recognition by interacting with the R-2 group from the five/six-membered ring of β-lactams (32). While many groups interpret the observed increases of the K_m values between metallo-β-lactamase and its substrates to mean that Asn233 functions in substrate binding, it has yet to be shown that K_m is an accurate indicator of substrate affinity. In fact, pre-steady-state studies of the subclass B3 L1 metallo-β-lactamase from the Stenotrophomonas maltophilia enzyme suggest that the substrate binding constant (K_s) and K_m values differ for nitrocefin (11). Instead, it was observed that substitutions at position 233 affect intermediate formation during catalysis (11, 42). Therefore, we are hesitant to suggest that the observed increases in K_m indicate weaker substrate binding but that perhaps it alters the constants from which K_m is derived. For example, the changes in K_m could also be the result of differences in the rate constants for substrate activation leading to intermediate formation.

Molecular modeling of substrates into the active site of the subclass B1 and B2 enzymes suggests that a direct interaction of the Asn233 side chain amine with the carboxylate moiety of β-lactam substrates can occur (13, 34, 36, 44, 46). This interaction was proposed to stabilize transition-state intermediates by contributing to the oxyanion hole together with Zn1 and Lys224 (13). The steady-state kinetic parameters of the mutant enzymes suggest that a role for the Asn233 side chain in the oxyanion hole is unlikely, as many substitutions increase rather than decrease the turnover number (k_cat). Substitution of the Asn233 residue could modify the catalytic mechanism by an alternate as-yet-unclear mechanism (35).

Conclusion.

Knowledge of the mechanisms of substrate recognition and catalysis in metallo-β-lactamases remains limited. This information is necessary to aid in the design of novel β-lactamase inhibitors. Asn233 is conserved among metallo-β-lactamases, and its role in enzyme function has been a subject of speculation (11, 13, 16, 20). In this study, the 19 variants at position 233 were characterized with respect to antibiotic resistance, in vivo protein expression levels, and, for a subset of mutants, kinetic parameters for hydrolysis of different classes of β-lactams. These studies revealed that many substitutions provide wild-type levels of antibiotic resistance toward these medically relevant drug substrates. An interesting question is why, if multiple substitutions function at position 233, is asparagine broadly conserved at this position among each of the three subclasses of class B β-lactamases (Fig. 5)? The origin or reservoir of metallo-β-lactamases and their natural substrates remains unclear (37). It is possible that a naturally occurring β-lactam molecule for which there are stringent sequence requirements for metallo-β-lactamase function shaped the evolution of the class B enzymes, resulting in the conservation of asparagine at position 233. For example, as seen in Table 1, imipenem hydrolysis imposes the most stringent sequence requirements on IMP-1 position 233. Thus, a naturally occurring molecule, such as thienamycin, which the carbapenem antibiotics are based on, may have driven the conservation of asparagine at position 233 (10, 23). Further characterization of the substrate profile and sequence requirements of metallo-β-lactamases and other metallo-β-lactamase superfamily members may resolve these questions (2).