GlycoNet researchers have uncovered an important piece to the puzzle of how a specific class of complex enzymes found in many bacteria function. The discovery that these enzymes need certain carbohydrate structures to operate could be the key to developing non-antibiotic treatments and aiding efforts to prevent antimicrobial resistance. 

One of the challenges to understanding these enzymes has been their architectural complexity. “We managed to visualize the complete 1600 amino acid multimodular enzyme,” says Alisdair Boraston, GlycoNet researcher and professor at the University of Victoria. In comparison, a typical enzyme or protein might have only 300 or 400 amino acids.

The class of enzymes used in the initial structural study is quite common in bacterial pathogens and the one studied comes from a poultry pathogen, Clostridium perfringens, which attacks the intestine of birds. Strains of the same pathogen can affect humans, but it’s a large problem in the poultry industry. 

Examining the enzyme architecture revealed two main features. The first is that the enzyme recognizes and cleaves within the amino acid sequence (or peptide) of mucin, a component of the mucus layer surrounding the cells. The second feature is that the enzyme recognizes a specific carbohydrate structure in the mucin. Mucin is rich in carbohydrates (or glycans) that  help it act as a barrier, which the enzyme must “eat” through to get to the cell. What is surprising is the extent to which these complex enzymes have adapted to needing the glycans as part of their function. 

“This is the first group of enzymes for which both the peptide sequence and the carbohydrate structure have been shown to be required in order for degradative activity to happen,” explains Warren Wakarchuk, scientific director of GlycoNet and a professor at the University of Alberta.

 “What we’re trying to do now is link back to the question of why these bacteria want to make enzymes that are specifically targeting a carbohydrate structure on a mucin.” 

As Boraston and Wakarchuk continue to study this class of enzymes, they are interested in mapping out which carbohydrates are required by these enzymes in order to function. They have expanded the study to include approximately 30 different enzymes that are structurally related and play a role in host-bacteria interactions affecting the health of not only animals but humans as well. 

Applications in health and science

“The hope is that if we can understand how these enzymes target things, that we might be able to develop a non-antibiotic way of dealing with some of these pathogens,” says Wakarchuk.

“Because these enzymes end up outside the cell to do their job, we don’t need to kill the bacteria. We just need to stop the enzyme.”

In terms of a therapeutic target, the main focus in the enzyme would be the catalytic domain, which causes the damage. “If you inhibited the catalytic activity, that would completely destroy its function,” says Boraston. 

A more readily implemented application of the research involving these enzymes is in mass spectrometry, a method to identify the chemical makeup of a substance. The enzymes could be used to “chop up” glycoproteins and, through understanding the specificity of these enzymes, scientists can interpret the fragmentation patterns to determine the mucin and carbohydrate structure.

Partnering for success

While both Boraston and Wakarchuk have a similar scientific upbringing, they specialize in different niches needed to make this study possible. 

“I have microbiology training, I have knowledge in glycobiology and knowledge in enzymology. What I can’t do is make the substrates for these enzymes,” explains Boraston.

That’s where Wakarchuk comes in: “What’s always been of interest to me is to be able to tailor make a substrate.” 

“We’re expanding the list so that hopefully we can cover all the specificities of the different enzymes,” says Wakarchuk. 

“It’s been a really nice synergy.” 

The research has received funding from GlycoNet.

November 18-24 is World Antimicrobial Awareness Week, which aims to increase awareness of and promote best practices to prevent the spread of antimicrobial resistance.