Two significant challenges have hindered research efforts in this area: The five sugars strung together to compose this section of the molecule are difficult to chemically prepare in the lab, and one of the key enzymes that initiates the structure's growth process doesn't easily function in a water-based solution in a test tube.
Ohio State synthetic chemists and biochemists put their heads together to solve these two problems, Woodward said.
To produce the five-sugar chain, the researchers started with a chemically prepared building block containing a single sugar and introduced enzymes that generated a five-sugar unit from that single carbohydrate.
"The first part was done chemically, and in the second part, we used the exact same enzymes that are normally present in a bacterial cell to transform the single sugar into a five-sugar string," Woodward said.
Once these sugars join to make a five-sugar chain, a specific number of these chains are joined together to fully form the O-polysaccharide. A protein is required to connect those chains the protein that doesn't respond well to the test-tube environment.
Early attempts to produce this protein in the lab resulted in clumping structures that did not function. So Woodward and colleagues produced this protein in the presence of what are known as "chaperone" proteins.
"And basically what the chaperones do is help the protein fold into its correct state. We were able to produce the desired enzyme and also were able to verify that it was functional," Woodward said.
This protein is called Wzy. It is a sugar polymerase, or an enzyme that interacts with the five-sugar chain to begin the process of linking several five-sugar units together.
Getting this far into the process was important, but the researchers al
|Contact: Robert Woodward|
Ohio State University