Core Concepts
In this article, you will learn how β-lactam antibiotics like penicillin work on a biochemical level, what characterizes different forms of penicillin, and how penicillin reshaped the microbiology field.
The Discovery of Penicillin
Some of history’s most impactful medical discoveries arose by accident. In the early 20th century, bacterial disease was widespread, and biomedical scientists like Alexander Fleming devoted their research to understanding and fighting against these types of infections.
Fleming’s research of Staphylococcus, a genus of bacteria, required cultivating it on Petri dishes. During one instance in 1928, he left the Staphylococcus to grow on the dishes while he was traveling. After his vacation, he returned to an unusual observation in the laboratory. Penicillium fungus (mold) had partially contaminated one dish that already had bacterial growth. On that dish, the bacteria encircling the fungus had died, while other bacterial colonies farther away from the fungus on the same dish were thriving.
Without antibiotics present on the dish, bacteria would grow freely. But when bacteria grow on a dish that has antibiotic disks, the impact is visibly obvious. If the antibiotic is effective against this type of bacteria, then that antibiotic disk will kill the bacteria around it, resulting in a zone of inhibition. This zon`e is a region on the Petri dish that lacks bacterial growth because the bacteria there have been eradicated. If the antibiotic is present, but is not effective against this type of bacteria, then the bacteria can continue to grow next to the antibiotic disk without any zone of inhibition.
Based on this observation, Fleming concluded that the culture broth used in the Petri dish had a bacteria-killing (bactericidal) property, which he named penicillin. He also determined that the fungus was naturally producing the penicillin and secreting this compound into the broth. Through further studies, he showed that the broth was capable of killing only some bacterial species, while others were unaffected. Despite his dedicated efforts to purify penicillin, he was unsuccessful and instead shared the fungus with other researchers who later built upon his work.
How do β-lactam antibiotics work?
We now understand that penicillin is an antibiotic, a type of medicine that targets and kills some forms of pathogenic bacteria. Although Fleming’s research is the basis of what we know about penicillin, later studies by Edward Abraham, Ernst Chain, and Dorothy Hodgkin determined the structural components that underlie its antibiotic function. Let’s investigate these mechanisms behind penicillin’s bactericidal abilities.
The Biochemistry of Penicillin
Penicillin belongs to the β-lactam class of antibiotics, meaning that its molecular structure has a β-lactam ring. This ring features a cyclic amide whose nitrogen atom is bonded to a carbonyl group’s β-carbon atom.
In penicillin, this nitrogen atom is also part of a thiazolidine ring that’s adjacent to the β-lactam ring. This combined structure is known as a penam ring. Penicillin, in all of its different forms, always contains a penam ring. However, what distinguish unique forms of penicillin are the different R groups within their side chains.
Peptidoglycan
All but two types of bacteria (Mycoplasma and L-form bacteria) have a cell wall. The cell wall, in bacteria that have it, is reinforced by an outer membrane made of peptidoglycan. Each peptidoglycan monomer consists of two sugars: N-acetylmuramic acid (NAM), which is also attached to a tetrapeptide, and N-acetylglucosamine (NAG). The four peptides within the tetrapeptide are diverse across different bacterial species. Tetrapeptide chains of individual monomers cross-link to form a mesh-like crystal lattice around the exterior of the bacterial cell.
Peptidoglycans enclose the bacterial cell in a three-dimensional, scaffolding-like formation that maintains the cell’s structure. β-lactam antibiotics can more easily penetrate a thin peptidoglycan layer than a thick one, so bacterial cells with a thin peptidoglycan layer are easier to kill. After penicillin penetrates this layer, it sabotages the bacterial cell’s membrane from the inside. But how does it accomplish this?
Cells with a cell wall have turgor pressure (hydrostatic pressure), a force that presses the cell membrane against the cell wall, thereby maintaining the cell’s shape. This force arises from the osmotic gradient between the inside and outside of the cell. If an imbalance in the osmotic gradient causes too much water to enter the cell, as observed in hypotonic conditions, the cell may lyse (burst open) and die. To prevent lysis, there must be another force pressing inward on the cell wall, so as to counteract the turgor pressure’s outward force. This inward force is provided by the peptidoglycan layer. Because peptidoglycan reinforces the cell wall, bacterial cells with a thick peptidoglycan layer are more resilient against the threat of lysis.
How does penicillin target the peptidoglycan layer?
DD-transpeptidase is the critical enzyme that cross-links the tetrapeptides to synthesize the peptidoglycan layer. β-lactam antibiotics are effective against pathogenic bacteria because their β-lactam ring binds to DD-transpeptidase’s active site, inhibiting the enzyme and preventing peptidoglycan formation. Without peptidoglycan, the bacterial cell’s outer membrane weakens as the turgor pressure overcomes the inward force on the cell well. Eventually, this membrane is so weak that it can no longer maintain the concentration gradient between the inside and outside of the cell. Too much water enters the bacterial cell, causing cell death.
Consequently, β-lactam antibiotics like penicillin are ineffective in treating infections caused by bacteria that lack a cell wall. These bacteria won’t lyse as a result of an osmotic imbalance, so only classes of antibiotics that don’t target cell wall synthesis can kill them. Healthcare professionals sometimes need to use a combination of different antibiotics to successfully neutralize these complex bacteria.
How is penicillin synthesized?
There are many types of penicillin; you may already be familiar with some, like amoxicillin or ampicillin, shown below. The β-lactam ring is core to every form, but slight variations in the synthesis process will change the R group, resulting in different forms of penicillin. For example, when the R group is a benzyl group, we call this penicillin G, but just one additional oxygen atom distinguishes it as penicillin V instead. However, recall that the β-lactam ring is the group that actually inhibits the DD-transpeptidase. Other structural groups within the penicillin molecule, like the variable R group, do not actively bind to the enzyme.
So why do we need to create unique forms of penicillin? Penicillin is not one-size-fits-all; each form is successful in combating different bacteria.
Generally, penicillin is manufactured on a large scale by fermenting Penicillium fungus, which naturally produces β-lactam antibiotics like penicillins G and V. When the mold produces enough penicillin, scientists isolate the penicillin product out of the mold mixture. Alternatively, specific forms of penicillin (such as amoxicillin and ampicillin) are modified versions of these natural molecules. Once the product meets the pharmaceutical industry’s rigorous quality standards, it’s ready for use as a medication.
Biosynthesis of Penicillin V
Let’s analyze how Penicillium naturally synthesizes penicillin V. We begin with three amino acids: L-α-aminoadipic acid (α-AAA), L-cysteine, and D-valine. With the help of the enzyme ACV synthetase, these amino acids undergo a condensation reaction to form a tripeptide called δ-(L-α-aminoadipyl)-L-cysteine-D-valine (ACV tripeptide).
Next, through oxidation, the enzyme isopenicillin N synthase (IPN synthase) transforms ACV tripeptide into isopenicillin N. This step closes the β-lactam ring, which we know is crucial to penicillin’s bactericidal properties.
The last step introduces phenoxylacetyl-CoA and acyltransferase to the synthesis. These components carry out a transamidation reaction, in which the α-AAA portion of IPN separates from the rest of the molecule. CoA activates IPN’s carbonyl group, preparing it to accept CoA’s phenoxyacetyl group as a substituent. Finally, having donated its phenoxyacetyl group to the IPN, CoA departs from the reaction, leaving us with our target molecule, penicillin V.
This synthesis resulted in penicillin V, but chemists can further modify this mechanism to yield other forms of penicillin in the lab. When Penicillium makes penicillin G in nature, it follows this same pathway, but uses phenylacetyl-CoA instead (one fewer oxygen atom than the phenoxyacetyl-CoA reactant seen here). Using phenylacetyl-CoA is the only modification necessary to create penicillin G instead of penicillin V. In theory, any R group could replace the CoA’s phenylacetyl group or phenoxyacetyl group in order to craft a different form of penicillin.
Penicillin V Synthesis: Semisynthetic Route
Semisynthesis is a synthesis approach where the starting materials are compounds found in nature. This serves as a shortcut for synthetic chemists: rather than producing the starting materials themselves, all they need to do is isolate the starting materials from wherever they naturally originate (for instance, from cell cultures).
To make penicillin V semisynthetically, we’ll use penicillin G as the starting material. Remember, this is one of the forms that Penicillium produces naturally. An enzyme called penicillin G acylase facilitates a straightforward hydrolysis reaction which yields two intermediates: 6-aminopenicillanic acid (6-APA) and phenylacetic acid. The water molecule used for hydrolysis gets split into a hydrogen atom and a hydroxyl group, which bond to the 6-APA and the phenylacetic acid, respectively.
The phenylacetic acid is not relevant to the next step in the synthesis, so we will disregard it at this stage. Instead, note how the 6-APA intermediate’s structure resembles that of a β-lactam antibiotic. We combine the 6-APA with a new reactant, phenoxyacetic acid. The 6-APA loses one hydrogen atom from its primary amine, and phenoxyacetic acid loses its terminal hydroxyl group. The equivalent of one water molecule leaves during this step, and the reaction prepares the fully-formed penicillin V molecule.
Incorporating 6-APA has strong advantages in the context of antibiotic synthesis. As we can see, it only takes one simple step to transform 6-APA into the desired penicillin product. This makes 6-APA a useful and dynamic intermediate when synthesizing any β-lactam antibiotic. Replacing the phenoxyacetic acid with a carboxylic acid that has a different side chain would yield a form of penicillin that has a different R group. In this way, compared to nature’s biosynthesis, semisynthesis is a springboard for faster, simpler, more versatile penicillin production. It’s also a viable option for making antibiotics that are never present in nature, like amoxicillin and ampicillin.
Penicillin and Antibiotic Resistance
Antibiotics like penicillin exist in a delicate balance. They can destroy enough bacterial cells to cure an infection, but they can’t kill every single bacterial cell. The antibiotics kill the weakest cells which are most susceptible to attack; therefore, the surviving bacterial cells are the strongest ones. These strong cells can still cause an infection and reproduce, and because this strength is an advantageous adaptation, it often gets passed down to their daughter cells too. Eventually, this population of bacteria consists predominantly of cells that can withstand an antibiotic treatment.
This adaptation presents a medical dilemma: antibiotics can cure devastating diseases, but can also give rise to bacteria that are so strong that the antibiotics can’t kill them. These strong bacteria are resistant to antibiotics. A major contemporary challenge is how to use antibiotics in a manner that maximizes their effectiveness while minimizing antibiotic resistance. This is why it’s important to have many different forms of penicillin: the more varieties of antibiotics available, the more likely it is that at least one of them will be effective.
Fortunately, the search for new antibiotics is ongoing. Some of them could prove to be effective options for treating infections caused by resistant bacteria. How do we identify new antibiotics? Some are found unintentionally (as Fleming discovered penicillin). Other antibiotic research involves isolating microbes from soil and screening their compounds for antibacterial activity. Alternatively, biopharmaceutical scientists use synthesis strategies to modify known antibiotics in an attempt to create a new one. All of these efforts drive the end goal of providing cures for currently-untreatable bacterial infections.
How does antibiotic resistance work?
To be resistant to antibiotics, a bacterial cell must have some property that invalidates or neutralizes the antibiotic’s attack mechanism. Let’s investigate the mechanisms behind β-lactam antibiotic resistance as an example.
B-lactamase
As we know, the β-lactam ring is responsible for killing bacteria by preventing the formation of the peptidoglycan layer. Anaerobic bacteria can produce β-lactamase, an enzyme that hydrolyzes the β-lactam ring’s amide bond. B-lactam rings are susceptible to hydrolysis because they have a lot of ring strain, which makes them quite reactive. Without an intact β-lactam ring, the antibiotic can’t target the bacterial cell wall. In this case, even if antibiotics are administered, the bacteria will be unaffected by them.
However, β-lactamase inhibitors can be co-administered alongside the antibiotics. The powerful combination of a β-lactam antibiotic and a β-lactamase inhibitor is more likely to defeat resistant bacteria compared to using the antibiotic alone. B-lactamase inhibitors serve as antagonists by irreversibly binding to and inactivating β-lactamase. Now that β-lactamase isn’t hindering the antibiotic’s β-lactam ring, the antibiotic can work normally. Examples of widely available β-lactamase inhibitors include clavulanic acid and sulbactam.
Penicillin-Binding Proteins
Penicillin-binding proteins (PBPs) catalyze reactions that are responsible for forming the peptidoglycan layer. Therefore, inhibiting PBPs deforms or weakens the bacterial cell wall, and ultimately leads to lysis. Recall DD-transpeptidase, the enzyme that creates the cross-linkages in peptidoglycan: this is one PBP.
A PBP can bind to penicillin at its β-lactam ring’s amide bond, forcing the ring to open, like β-lactamase does. This bonding irreversibly inactivates the PBP and renders the penicillin ineffective. One notable example of a bacterium that expresses PBPs is methicillin-resistant Staphylococcus aureus (MRSA), where methicillin is a form of penicillin.
As such, PBPs are another research focus among scientists who are striving to resolve antibiotic resistance. Overcoming PBPs would prevent them from synthesizing peptidoglycan and make it easier for penicillin to attack the bacterial cell. Recent research explores how modifying a PBP’s structure, specifically the amino acids within it, influences how penicillin binds with PBPs. If a PBP is altered such that it can’t bind to penicillin’s β-lactam ring, the PBP will not interfere with penicillin’s antibiotic function.
Conclusion
Alexander Fleming’s unexpected discovery of penicillin changed the course of microbiology research. B-lactam antibiotics use their β-lactam ring to inhibit DD-transpeptidase and weaken a bacterial cell’s peptidoglycan-based cell wall. There are numerous variations of penicillin, as well as many possible ways to produce them, including biosynthesis and semisynthesis.