Heparosan-Glucuronate 5-Epimerase Enzyme Substrate Specificity
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current biosynthesis pathways of heparosan and heparin do not fully elucidate the enzymatic cascade in Achatina fulica, particularly the substrate specificity and product identification of IdoA synthase, and no protein with homology to heparosan-N-sulfate-glucuronate 5-epimerase has been identified in Achatina fulica.
Innovation Solution
A novel heparosan-glucuronate 5-epimerase (HG-5epi) is isolated from Achatina fulica, which epimerizes glucuronic acid residues to iduronic acid in N-acetyl heparosan and vice versa in completely desulfated N-acetylated heparin, with specific substrate specificity different from known C5-epimerases, and methods for producing this enzyme and polysaccharides with epimerized hexuronic acid residues are developed.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If known C5-epimerases are used for biosynthesis of heparin and heparan sulfate, then they can epimerize GlcA residues to IdoA residues in N-deacetylated N-sulfated heparosan (NSH), but they cannot act on N-acetyl heparosan (NAH) itself
Solution Approach 1:
The patent applies parameter changes by modifying the substrate acceptance parameters of the epimerase enzyme. The discovered HG-5epi enzyme has different substrate specificity parameters compared to known C5-epimerases, allowing it to accept NAH (N-acetyl heparosan) as substrate whereas known enzymes only accept NSH (N-deacetylated N-sulfated heparosan). This parameter change in substrate recognition enables a simplified biosynthetic pathway.
2Ease of manufacture
If the biosynthesis pathway uses multiple enzymatic steps including N-deacetylation and N-sulfation before epimerization, then known C5-epimerases can function, but the pathway becomes more complex and the enzymatic cascade is not fully elucidated
Solution Approach 1:
The patent applies preliminary action by enabling epimerization to occur earlier in the biosynthetic pathway. The HG-5epi enzyme can perform epimerization on NAH before N-deacetylation and N-sulfation steps, whereas known C5-epimerases require these modifications to occur first. This reordering of enzymatic actions simplifies the overall pathway and fills the gap in understanding the complete enzymatic cascade in Achatina fulica.
3Adaptability or versatility
If Achatina fulica produces polysaccharides with varying iduronic acid content, then diverse physiological activities can be achieved, but control over specific iduronic acid content is limited
Solution Approach 1:
The patent applies feedback control through the use of HG-5epi enzyme which enables precise control over iduronic acid content in produced polysaccharides. By utilizing this enzyme with its specific substrate specificity, the biosynthetic pathway can be regulated to produce polysaccharides with predetermined iduronic acid concentrations, achieving manufacturing precision while maintaining the ability to produce diverse physiological activities.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The HG-5epi enzyme enables the production of polysaccharides with controlled iduronic acid content, expanding the understanding of biosynthesis pathways and providing novel materials with potential physiological activities similar to heparin and heparan sulfate.
Implementation Method 1
a heparosan-glucuronate 5-epimerase (HG-5epi), which epimerizes glucuronic acid residues to iduronic acid in N-acetyl heparosan and vice versa in completely desulfated N-acetylated heparin
Data Source
Figure 1~2
Figure 3~4
Figure 5~6
AI summary
To provide a polypeptide having heparosan-glucuronate 5-epimerase activity, whereby means for producing a polysaccharide in which hexuronic acid residues has been epimerized is provided. Through screening of Achatina fulica cDNA library, a DNA encoding a polypeptide of heparosan-glucuronate 5-epimerase is obtained. The epimerase acts on glucuronic acid residues of N-acetyl heparosan and/or iduronic acid residues of completely desulfated N-acetylated heparin. The polypeptide encoded by the DNA is expressed by insect cells, to thereby yield the polypeptide having heparosan-glucuronate 5-epimerase activity. By bringing the polypeptide into contact with N-acetyl heparosan or completely desulfated N-acetylated heparin, a polysaccharide in which hexuronic acid residues has been epimerized is yielded.