Sandbox Reserved 994

This is a default text for your page '. Click above on edit this page' to modify. Be careful with the < and > signs.

You may include any references to papers as in: the use of JSmol in Proteopedia ^[1] or to the article describing Jmol ^[2] to the rescue.

Background

Bacteria have cell walls which are largely made of a material called peptidoglycan, composed of NAM and NAG, which the cell uses to protect against lysis from changing osmotic pressure. These two materials make alternating links of NAM and NAG which are held next to each other by NAM cross links. Without this forming, the bacterial cell wall cannot hold its structure against more hypotonic solutions and the cell will lyse and die. Beta-lactams are a group of antibiotics that block the NAM cross linking in the bacterial cell wall by “irreversibly binding to the enzymes that cross-link NAM subunits.^[3]” Beta-lactams are a family of antibiotics that have a four membered ring called a beta-lactam ring. To answer for this, bacterial cells have developed enzymes called beta-lactamases that will hydrolyze the bond between the ring’s acyl group and its nitrogen.^[4]

Acinetobacter baumannii is a strain of bacteria which is resistant to a large amount of β-lactams and produces three different types of β-lactamases; it was also involved in a nosocomial outbreak in Spain.^[5] It is dangerous in hospitals because its antibacterial resistance allows it to infect patients who are taking antibiotics. By taking antibiotics, they are clearing out their system and clearing the way for this bacteria to take over.^[6] This strain of bacteria uses three different β-lactamases to protect itself from antibiotics, one of which is a newly discovered enzyme called OXA-24 β-lactamase. This enzyme is inhibited by chloride ions, tazobactam, sulbactam, and clavulanic acid; and specifically counteracts benzylpenicillin and cephaloridine.^[5]

OXA-24 is a member of the carbapenem-hydrolyzing class D β-lactamases (CHDLs) and is expressed as a resistance mechanism by the bacteria Acinetobacter baumannii. Class D β-lactamases are clinically dangerous because they hydrolyze β-lactam antibiotics such as penicillins, cephalosporins, and carbapenems. Class D β-lactamases are classified as OXA’s, which is in reference to their class designation as oxacillinases; however the terminology is somewhat misleading. While they do have very strong affinity for the antibiotic oxacillin^[7]; the OXA’s have expanded since their discovery to include penillinase, cephalosporinase, and carbapenemase activity in their spectrum. Due to their original designation as oxacillinases, however, the assignment of the prefix OXA has continued to be standard designation.

Bacterial Resistance

Since the discovery of penicillin by Alexander Flemming in 1928, antibiotics have revolutionized the medical world. Penicillin is known as a β-lactam antibiotic, which is characterized by a four-membered β-lactam ring (a cyclic amide). There are four classes of β-lactam antibiotics: monobactams, which are the simplest class of β-lactam and aren’t fused to any rings; penicillins, which have a thiazole ring fused to the β-lactam; cephalosporins, which contain a thiazine ring; and lastly carbapenems, which are fused with a pyrrole ring and are considered a last line of defense. ^[8] β-lactam antibiotics are the most widely used class of antibiotics because they successfully fight most bacterial infections by inhibiting cell wall synthesis. Their mechanism of action is through inhibition of the transpeptidas enzymes, which are located in the bacterial cell membrane. Transpeptidase is alternatively referred to as a penicillin-binding protein (PBP) and is responsible for catalyzing the cross-linking of the bacterial cell wall ^[9]. β-lactams mimic the structure of the terminal D-alanine chain of peptidoglycan and irreversibly bind to PBP, disrupting the cross-linking process that is critical to cell wall synthesis. As a result, the bacterial cell wall is compromised and the bacteria lyse and die.^[10]

Due to overperscription and misuse of antibiotics, bacteria have been able to develop resistance mechanisms. One of these resistance mechanisms is through the expression of β-lactamases, which have evolved as a seperate enzyme over millions of years from PBP.^[11] β-lactamases act by hydrolyzing the β-lactam ring, which renders the antibiotic inactive before it has a chance to inhibit the transpeptidase enzymes.^[12] β-lactamases are grouped into four different classes (A, B, C and D); all of which, with the exception of class B, use a serine based mechanism for destruction of β-lactams. Class B β-lactamases use zinc ions for hydrolysis. Class D was distinguished from other serine β-lactamases in the late 1980s, due to having an affinity for oxacillin as its substrate in addition to other antibiotics.^[13] Even more concerning is that the class D β-lactamases, or OXAs, are not inhibited by current clinical β-lactamase inhibitors such as clavulanic acid. OXA-24, which has considerable carbapenemase activity, poses a dangerous clinical threat due to the absence of an effective inhibitor.

Structure

OXA-24 is a monomeric protein with an active site composed of a short α-helix and a β-sheet. The active site of OXA-24 is characterized by a hydrophobic pocket, which is representative of Class D β-lactamases as a whole. The hydrophobic bridge contributes to the substrate specificity for carbapenems and is composed of an arrangement of the Tyr-112 and Met-223 side chains.^[14] These residues block the active site and only allow a very specific binding configuration of antibiotics. The active site is overall positively charged and contains a sulfate ion along with other solvent molecules when no substrate is bound. The mechanism of attack is through the use of three catalytic residues: Serine-81, Carboxylated Lysine-84, and Serine-128. The hydroxyl chain of Ser-128 conforms in the direction of the active-serine Ser-81, and contributes to the catalytic mechanism.^[14]

Hydrolysis Mechanism

β-lactam antibiotics (basic structure of a β-lactam is shown above) are hydrolyzed by β-lactamase enzymes, utilizing a covalent catalysis serine-based mechanism. The β-lactamase cleaves the amide bond of the four membered ring which renders the antibiotic inactive before it reaches its bacterial target, the transpeptidase enzymes.

The mechanism of attack involves a catalytic serine residue, a carboxylated lysine, and another active site serine which contributes to proton movement (A). A high energy tetrahedral intermediate (B) is generated and an acyl enzyme intermediate (C) is formed after the cleavage of the four-membered ring. KCX84 activates the deacylating water which completes the reaction leaving a hydrolyzed β-lactam ring and a regenerated β-lactamase.^[7]

^[15]

Catalytic Activity

There are three catalytic residues involved in the hydrolysis of β-lactam antibiotics. Serine-81 is the catalytic serine, which performs a nucleophilic attack on the β-lactam ring after being deprotonated by the carboxylated lysine-84 (KCX84). This carboxylated lysine is formed by CO2 in the environment engaging in unfavorable interactions with the hydrophobic pocket of OXA-24, so it carboxylates Lysine-84 (A).^[7]

The second step (B) forms a high energy intermediate. The cyclic amide from the β-lactam deprotonates Serine-128 (S128), which proceeds to deprotonate the amine on KCX84, which deprotonates the carboxylate group. This high energy intermediate resolves to form the stable acyl-enzyme intermediate.^[7]

The third step (C) proceeds with the use of a catalytic water, which is deprotonated by KCX84. The water now can mount a nucleophilic attack on the ester linkage connecting S81 and the hydrolyzed β-lactam. This forms a high energy dissociation intermediate, where S81 is released by a mechanism, which has not quite yet been determined. It is suspected that it deprotonates KCX84, but this has not yet been confirmed.^[7]

In step four (D), The enzyme is successfully regenerated and the hydrolyzed β-lactam antibiotic is released back into solution. OXA-24 is now free to hydrolyze another substrate and the antibiotic has been rendered useless.^[7]

This is a sample scene created with SAT to by Group, and another to make of the protein. You can make your own scenes on SAT starting from scratch or by loading and editing one of these sample scenes.