Structures of the Michaelis-Complex (1.2 Å) and the Covalent Acyl-Intermediate (2.0 Å) of Cefamandole Bound in the Active Sites of the Mycobacterium tuberculosis β-Lactamase K73A and E166A Mutants

Abstract

The genome of Mycobacterium tuberculosis contains a gene that encodes a highly active β-lactamase, BlaC that imparts TB with resistance to β-lactam chemotherapy. The structure of covalent BlaC-β-lactam complexes suggests that active site residues K73 and E166 are essential for acylation and deacylation, respectively. We have prepared the K73A and E166A mutant forms of BlaC and have determined the structures of the Michaelis-complex of cefamandole, as well as the covalently bound acyl-intermediate of cefamandole at resolutions of 1.2 and 2.0 Å, respectively. These structures provide insight into the details of the catalytic mechanism.

β-lactams are one of the most important classes of antibacterial chemotherapeutics in clinical use today. These compounds disrupt bacterial cell wall biosynthesis by irreversibly inhibiting the D,D-transpeptidases that are responsible for cross-linking the pentapeptide components of polymerized peptidoglycan precursors via specific peptide linkages. The cross-linked peptidoglycan stabilizes the bacterium against high internal osmotic pressure (1–3). β-lactams have proven ineffective in the treatment of infections by Mycobacterium tuuberculosis (TB), a pathogen infecting nearly one in three people across the globe (4). Mycobacterium tuberculosis harbors a genomically encoded extended-spectrum β-lactamase (ESBL), BlaC (5). BlaC has been shown to rapidly hydrolyze penicillin, cephalosporin, and carbapenem classes of β-lactams (6). BlaC and other β-lactamases bind β-lactams and form a covalent acyl-intermediate, like the D,D-transpeptidases, but have evolved an activity that results in the hydrolysis of the covalently bound product in the active site (7). As a result, β-lactamase catalysis effectively destroys the antibiotic, thwarting β-lactam chemotherapy.

β-lactamases have been categorized according to two different schemes. The Ambler system classifies these enzymes according to sequence homology as Ambler classes A through D (8), while more recently Bush, Jacoby and Medeiros have devised groups 1 through 4 based on substrate/inhibitor profiles (9). BlaC is a class A, group 2 ESBL. The class A β-lactamases are structurally conserved with the Penicillin Binding Protein (PBP) domain of the D,D-transpeptidases and are likely the result of divergent evolution (10). In recent years there has been a dramatic rise in the number of group 2 ESBL enzymes isolated from antibiotic resistant strains of bacteria resulting from the broad utilization of β-lactams (9, 11). In contrast to the more recent β-lactamase isozymes, the genomically encoded blaC gene product likely has ancient evolutionary origins dating back to the original divergence of the class A and class D β-lactamases (12). Episodic positive selective pressure has been placed throughout history on these two classes of β-lactamases causing significant deviations from the mutational molecular clock by which the phylogeny would normally be deduced. Yet it has been established that the serine-type β-lactamases have been evolving from before the divergence of Gram-negative and Gram-positive bacteria, implying they have had an active protective role for more than 2 billion years (9, 12).

The class A β-lactamases have been investigated for the last 70 years (13), and a common mechanism of catalysis has emerged. The catalytic mechanism of class A β-lactamases occurs in three steps: binding of the β-lactam to generate the Michaelis-complex, attack by the conserved serine nucleophile (Ser70 in BlaC) to form a ring-opened covalent acylated-enzyme intermediate and activation of a conserved active site water molecule for hydrolysis of the covalent acyl-intermediate from the enzyme allowing the hydrolyzed product to exit the active site. A number of previous studies have identified the active site of various β-lactamases bound with dead end inhibitors or mutant forms of the enzymes which have been able to `trap' the acyl-intermediate for observation (6, 14–21). In this study, we have employed mutational techniques to obtain high resolution crystallographic data of the BlaC active site bound with the second generation cephalosporin, cefamandole, in both the Michaelis-complex as well as the covalent-acyl intermediate, providing us with structural data relevant to the mechanism of the serine-type β-lactamase, BlaC.

The enzymatic mechanism of BlaC relies on two, highly conserved active site residues, Lys73 and Glu166, which are involved in the activation of the acylating nucleophile (Ser70) and the activation of the active site water molecule for deacylation (22, 23), respectively. Studies of the Glu166Ala mutant of BlaC have revealed that this residue is essential for deacylation, via hydrolysis by a hydrogen-bonded water molecule, of covalently acylated BlaC. It has been assumed that the ε-amino group of Lys73 in BlaC, by hydrogen bonding the Ser70 hydroxyl group, increases the nucleophilicity of the side chain hydroxyl, permitting attack at the β-lactam carbonyl-carbon. We have prepared and crystallized the K73A and E166A mutants of BlaC and have determined the structures of the Michaelis-complex of cefamandole (Figure 1A), as well as the covalently bound acyl-intermediate of cefamandole (Figure 1B) at resolutions of 1.2 and 2.0 Å, respectively (SI Table1).

Figure 1.

A) The 1.2 Å fo-fc omit density of the cefamandole (blue carbons) Michaelis-complex bound in the active site of BlaC-K73A (yellow carbons). B) The 2.0 Å fo-fc omit density of the cefamandole-BlaC-E166A covalent adduct (all blue carbons). These figures were produced in Pymol and contoured at 2 σ.

BlaC catalyzes the near diffusion-limited hydrolysis of many penicillins and cephalosporins, yet prior observations have associated the rate-limiting step with the second deacylation half reaction (6, 24). In the case of cefamandole, the turnover number of the wildtype BlaC enzyme is 3500 min⁻¹ with a k_cat/K_m value of 2 × 10⁷ M⁻¹ min⁻¹ (6). The K73A mutant exhibits a 2 × 10⁵ decrease in cefamandole activity with a k_cat/K_m value of 84 M⁻¹ min⁻¹, as well as more than a 10-fold increase in the K_m value making accurate k_cat measurements impossible due to the high absorbance. The slow reaction rate of BlaC-K73A allowed us to trap the Michaelis-complex of cefamandole, and determine the three-dimensional structure of the BlaC-K73A cefamandole complex before attack of the Ser70 nucleophile. This represents unambiguous evidence for the role of Lys73 as the primary activator of the Ser70 nucleophile in the first, acylation half reaction.

In the Michaelis complex, the cefamandole carboxylate group is hydrogen bonded by the side chain hydroxyl groups of Ser142, Thr253 and Thr251 at 2.4, 2.4 and 2.7 Å, respectively (Scheme 1A). In addition, there is a 2.9 Å hydrogen bond between the Thr253 backbone amide nitrogen and β-lactam carbonyl-oxygen, one of two interactions that stabilize the developing negative charge at the β-lactam carbonyl-oxygen. These initial contacts with the β-lactam carboxylate act as a hinge around which the β-lactam ring carbonyl-oxygen is swung into contact with the oxyanion hole, composed of the backbone amide nitrogens of both Thr253 and Ser70. Hydrogen bonding interactions also exist between the side chain hydroxyl of Ser70 and K73 (inferred from native and E166A structures), along with the catalytic water molecule and Glu166. The cefamandaole side chain exists in two conformations as evidenced by the clear electron density of the thioether sulfur.

Scheme 1.

A model of the active site of A) the cefamandole-BlaC-K73A Michaelis complex and B) the acyl-adductof cefamandole with the BlaC-E166A mutant. Implied residues and bonds from alternate BlaC structures are colored green.

When the cefamandole Michaelis-complex structure is compared with the acylated-adduct captured using BlaC-E166A (Scheme 1B), multiple changes suggest the order of catalytic events. First, the β-lactam ring carbonyl-oxygen is drawn deeper into the oxyanion hole allowing attack at the β-lactam carbonyl-carbon. While maintaining an initial contact distance of 2.9 –3.0 Å with the Thr253 backbone amide nitrogen, the β-lactam carbonyl oxygen shifts (from 3.1 Å to 2.7 Å) closer to the Ser70 backbone amide. The effect of being drawn further into the oxyanion hole is that the position of the β-lactam ring carbonyl-carbon moves from a distance of 2.8 Å from the Ser70 hydroxyl oxygen atom to form a covalent bond (1.3 Å) with the hydroxyl group, generating the ester bond and opening the β-lactam ring. The Ser70 hydroxyl side chain position appears unchanged between the WT apo, Michaelis complex and acylated covalent intermediate, indicating that the β-lactam ring moves into contact with a rigid Ser70 side chain. In concert with these movements, the cefamandole carboxylate shifts from the initial binding positions of 2.4 to 2.9 Å with the Thr253 hydroxyl and from 2.7 to 2.5 Å with the Thr251 hydroxyl, while the hydrogen bond with the Ser142 side chain hydroxyl is broken. In related structures of carbapenem β-lactams covalently bound to BlaC, β-lactam ring opening is accompanied by a tautomerization of the α,β-unsaturated eneamine to the corresponding imine, with protonation at the side chain (15). In this case, the 1,3-thiazine ring generated upon cefamandole ring opening retains the carbon-carbon double bond of the eneamine. This may be due to the observation that as the 1,3-thiazine ring nitrogen is protonated, the Ser142 hydroxyl exchanges its hydrogen bond from the carboxyl group and flips nearly 70 degrees to hydrogen bond to the newly formed secondary amine. This active site rearrangement, along with decreases in the hydrogen bonding distance between the cefamandole R group secondary hydroxyl and the carboxamide side chain of Asn186, helps to stabilize the covalently bound cefamandole intermediate (25).

Essential to the deacylating second half reaction is the conserved active site water. In the first half reaction, Lys73 gains a proton upon deprotonation of the Ser70 hydroxyl. This likely causes a change in the pK value of the neighboring Glu166 sidechain, generating the general base required for the activation of the water molecule and deacylation. This water is also tightly associated with Asn186, which together with Glu166 serve to orient the water molecule and activate it as a nucleophile. Prior to the formation of the acyl-intermediate the activated water is positioned 3.4 Å from the β-lactam ring carbonyl-carbon. After the attack by Ser70 and the formation of the covalent ester bond, the carbonyl-carbon is an estimated 2.1 Å from the activated water (as determined by superposition of the WT apo BlaC catalytic water position). The estimated water distances of 2.6 Å from E166 and 2.1 Å from the carbon would allow for rapid and efficient deprotonation and “hydroxide” attack. The partial positive charge on the ester carbonyl, due to the influence of the oxyanion hole residues, and the alignment of the water/hydroxide molecule would allow for efficient attack. Finally the protonated ε-amino group of Lys73 in close proximity to the Ser70 hydroxyl side chain could assist in the decomposition of the tetrahedral intermediate and deacylation.

For the first time the Michaelis-complex of a β-lactam with a β-lactamase has been directly visualized using X-ray crystallography. The Michaelis-complex structure of cefamandole in conjunction with the ring-opened acylated intermediate of cefamandole bound within the active site, has given us insights into the BlaC active site and interactions that allow for the rapid acylation and deacylation reactions with this substrate. This information is currently being incorporated into the design of inhibitors.