Structural Basis of Antibiotic Resistance: Exploring β-Lactamases
Betsy M. Martinez-Vaz and Melanie Van Stry
Developed as part of the CREST Project funded by NSF-DUE #1725940; last revision 12-2020

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Exploring β-Lactamases

TEM-1 β-Lactamase

β-lactams are an important class of antibiotics that work by inhibiting cell wall synthesis in bacterial cells. This group of antibiotics is commonly used due to their low toxicity, availability, and affordable cost. β-lactams have the same active site and the same general structure– the β-lactam ring (fig. 1). This structure, the β-lactam ring, is effective in killing bacteria. This chemical structure competes with the D-ala-D-ala peptide that is used in the cross linking step in peptidoglycan biosynthesis. The transpeptidase enzyme that cross links the D-ala-D-ala binds to the antibiotic instead and is rendered ineffective, therefore, compromising the cross linking step in cell wall synthesis (video 1).

Video 1- β-Lactams: Mode of Action and Mechanism of Resistance

Despite the diversity and effectiveness of β-lactam antibiotics, bacteria have developed resistance to these drugs. There are various mechanisms bacteria employ to resist β-lactam antibiotics, however, the most common strategy is the production of β-lactamases enzymes. These proteins cleave the β-lactam ring rendering the antibiotic ineffective.

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Figure 1- Example structures of several classes of β-lactam antibiotics. The β-lactam ring is highlighted in red. Image adapted from PharmWiki at Tulane University.

TEM-1 is the most common extended spectrum β-lactamase present in Gram negative bacteria. This protein confers resistance to a variety of β-lactams antibiotics including ampicillin, penicillin and several cephalosporins.

See the overall 3D structure of TEM-1

Describe the elements of secondary structure present in TEM-1.

Exploring the Active Site of TEM-1

Describe the tertiary structure of TEM-1. Does this protein have quaternary structure?

List the amino acids shown in the active site of TEM-1.

Briefly describe the size and biochemical properties of the amino acids present in the active site of TEM-1. Are they polar, nonpolar, acidic, basic, etc?

Structure of Benzylpenicillin

Identify the β-lactam ring in Benzylpenicillin. Where in this molecule could Ser70 carry out a nucleophilic attack to break the β-lactam ring?

TEM-1 is a classified as a serine enzyme because it uses Ser70 as nucleophile to attack the carbon in the β-lactam ring of antibiotics. Benzylpenicillin is one of the substrates of TEM-1. When this drug binds the active site of TEM-1 the β-lactam ring is positioned near Ser70 through hydrogen bonding involving various amino acids residues. Let's take a look at the catalytic mechanism of TEM-1. Then, we will explore the the chemical structure of Benzylpenicillin and the acyl-enzyme reaction intermediate formed during catalysis.

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Catalytic Mechanism of TEM-1. (A) The acylation mechanism of Class A β-lactamases, Step 1: The nucleophilic attack and formation of the tetrahedral intermediate; Step 2: Formation of the acylenzyme from the tetrahedral intermediate (B) The deacylation mechanism of Class A β-lactamases, Step 1: The nucleophilic attack by a water molecule and formation of the tetrahedral intermediate; Step 2: The bond-breaking between Ser70-O of the enzyme and the β-lactam carbonyl carbon atom; Step 3: Hydrogen atom transfers from Glu166 to Ser70-O and the regeneration of protonation state of the enzyme. (Tao, et. al. 2019).

When Benzylpenicillin binds to the active site of TEM-1, the β-lactam ring is positioned near Ser70. Binding facilitates the nucleophilic attack on the ring leading to cleavage and inactivation of the drug. Let's take a look at benzylpenicillin bound to the active site of TEM-1. Notice the structure of benzylpenicillin. Can you see the β-lactam ring? Why?

This image shows the open structure of benzylpenicillin after nucleophilic attack by ser70. Notice the acylenzyme intermediate between carbon that used to be part of the β-lactam ring and the oxygen atom of the Ser70.

Benzylpenicilllin Intermediate bound to TEM-1

NDM-1 β-Lactamase

NDM-1 is present in multi drug resistant bacteria, called superbugs. It can inactivate many β-lactam antibiotics including penicillins, cephalosporins, and carbapenams by hydrolyzing the β-lactam ring. NDM-1 has a broad, hollow active site that accommodate different classes of β-lactams. The enzyme uses a modified catalytic triad with a Zn ion instead of a serine residue. Here we will explore the structure of NDM-1 bound to different antibiotics.

NDM-1 Structure

How many subunits are present in NDM-1?

These Jmol animations zoom in on the active site in one of the chains, showing the two Zn ions in orange and the amino acid side chains in CPK.

Which amino acid side chains coordinate the Zn ions?

Comparison of the reaction mechanisms for TEM-1 (A) and di Zn II Class B1 β-lactamases (B).

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From Meini et al. 2015

How does the reaction mechanism for TEM-1 differ from Class B1 metallo lactamases?

NDM-1 bound to Benzylpenicillin intermediate
NDM-1 bound to Meropenem intermediate
NDM-1 bound to Cefuroxime intermediate

How does the active site of NDM-1 compare to that of TEM-1?

Why is NDM-1 able to hydrolyze many different types of β-lactams?

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