New Delhi metallo-β-lactamase 1

Introduction

Antibiotic resistance

Microorganisms are becoming increasingly resistant to various antibiotics, which has caused global concerns in the field of medicine. Bacterial enzymes are largely responsible for the emergence of resistance, which play an active role in diverse biochemical processes^[1]. The primary biochemical mechanism for resistance; however, is supported by the gradual evolution of bacterial enzymatic superfamilies, because of the variations of the genetic code^[1]. Evolutionary modifications of bacterial enzymes play a significant role in impacting the success rate of bacterial antibiotics.

β-lactamases

Beta-lactamases are a class of bacterial enzymes responsible for the hydrolysis of the amide bond, located in the inner portion of the β-lactam ring, resulting in the greatly reduced activity of the β-lactam antibiotic^[2]. The β-lactam ring consists of the functional component of various β-lactam antibiotics, including cephalosporins and carbapenems^[1]. β-lactamases are further organized into four groups: classes A, C, and D are serine hydrolases and class B are metalloenzymes, or metallo-β-lactamases (MBLs)^[1][2]. Resistant enzymes have the capability to break down the β lactam ring, disrupting the efficiency of the antibiotics.

Background

Historical discovery

belongs to the class B MBL group of β-lactamases, most noted for its association with the Klebsiella pneumoniae pathogen. K. pneumoniae, a gram-negative bacterium, is a causative agent of a urinary and respiratory tract and bloodstream infections, specifically targeting patients with immunodeficiency^[3].

NDM-1 was first detected in the Klebsiella pneumoniae bacterium in 2009, from a urine culture from a Swedish patient, who traveled from India^[4]. Scientists soon discovered traces of its origin from New Delhi, the capital of India, hence giving the enzyme its name. Thirteen alternative forms of NDM were subsequently discovered, although, NDM-1 is the most common form^[4]. Although most of the microbial cultures originated from India, many other countries have been impacted globally by NDM since its discovery.

Structure

NDM-1 can be viewed as a hybridized structure, containing distinctive structural elements, as well as enzymes under the same classification system as the B class. NDM-1, a single chain polypeptide, is illustrated by an “sandwich” motif, which is characteristic of the MBL class^[5]. Furthermore, NDM-1 contains an additional β-strand augmentation located at the N terminus; this is accompanied by a β-turn motif^[5]. This differentiates the structure from enzymes VIM-2 and IMP-1, other enzymes of the MBL class.

Crystal structure

There has been supporting evidence showing crystalline growth for NDM-1, through investigation using fundamental laboratory techniques. Crystals of suitable quality were grown, which resulted in a solution to the structure that was solved at 1.9 Å resolution^[2]. The structure contains traces of cobalt (1.6052 Å), nickel (1.4881 Å), and zinc (1.2828 Å)^[2]. This supports the conclusion of the presence of a metal ion in the crystalline structure. The solvent content for the crystal structure is estimated to be around 53.6%^[5].

Active Site

NDM-1 contains two active sites. The first consists of one water molecule attached to ^[2]. The second active site has three different types of residues: , also containing water molecules^[2].

Understanding the basic “sandwich” motif and crystal structure is key to understanding the important conformational changes that act around the active site. The active site of NDM is located on loop L3, which stretches and deforms in the unbound form; however, there is an expanded β-sheet interaction in the presence of ligand binding^[5]. This creates a “zippering effect,” which pulls the L3 loop away from the NDM-1 zinc center, at an increasing distance. In response to this, the side chain M67 faces away from this center, instead, forming a hydrophobic reaction with an R1 ampicillin group^[5]. On the other hand, L65 travels toward the center^[5]. The result is an R1 phenyl group, comprised of L65, M67, and W93 residues, stabilized by hydrophobic interactions^[5]. This results in a more flexible structure, which may potentially impact its likeliness to react with β-lactam antibiotics.

After ligand-binding interactions, the N220 component of nitrogen moves closer to the zinc center, allowing for interaction with the lactam carbonyl group^[5]. Furthermore, the interaction with N220 and zinc-1 creates an oxyanion hole^[5]. The L3 and L10 loops allow for the flexibility of various binding-substrates, as well as with differing biochemical structures.

Protein expression

Research indicates there is an expression of a Type II lipidation signal peptide, on the N terminus^[5]. Studies show that there is a cleavage site between the C26 and M27 residues^[5].

Research applications

The emerging resistance of antibiotics because of various bacterial enzymes has resulted in increased concerns in the science community. Further research is currently in progress to investigate possible inhibitors for enzymes, such as β-lactamase^[1]. Some potential inhibitory candidates for resistance of class A β-lactamase enzymes include β-lactams, featuring products such as clavulanic acid, and sulbactam^[1]. Interestingly, there are inhibitors that contain minimal structural differences to that of β-lactams, lacking a β-lactam ring^[1]. These inhibitors have the possibility of forming carbonyl-enzyme complexes; these tests have been successful against A, C, and D classes^[1]. This leaves further investigations to find inhibitors for class B MBLs.

X-ray structures show that NDM-1 displays three different states: metal free, singly metalated, and doubly metalated. This observation can potentially provide more clues about the binding mechanism of NDM-1, providing noteworthy steps in catalysis, involving the recognition of the ligand sites ^[6]. In response, this would most likely increase the activation center for nucleophilic attack^[6].

Conclusion

Although β-lactamase, as well as class B MBL, has been widely studied, New Delhi metallo-β-lactamase 1 is still considered a recent evolutionary development. Although K. pneumoniae is not severely prevalent right now, it is a key example of a bacterial pathogen that has the capability of becoming resistant to β-lactam antibiotics. Analyzing the structure gives scientists further insight as to future topics for study and research. This involves both previous knowledge about broad-spectrum antibiotics and its reaction to bacterial enzymes, such as β-lactamase.