Glucuronoxylan recognition by GH 30 xylanases: A study with enzyme and substrate variants

Area of Science:

• Biochemistry and Enzymology
• Glycoside Hydrolase Family 30

Background:

• Erwinia chrysanthemi XynA (EcXyn30A) is a glycoside hydrolase family 30 enzyme.
• EcXyn30A specifically hydrolyzes 4-O-methylglucuronoxylan (GX).
• The carboxyl group of 4-O-methylglucuronic acid is a key substrate recognition element, interacting with Arg293.

Purpose of the Study:

• To investigate the role of Arg293 in EcXyn30A's substrate recognition and catalytic efficiency.
• To compare the kinetic parameters of wild-type EcXyn30A with an Arg293 to Alanine (R293A) variant using GX, its methyl ester (GXE), and reduced form (GXR).

Main Methods:

• Determination of kinetic parameters (kcat) for wild-type EcXyn30A and the R293A variant on GX, GXE, and GXR.
• Analysis of specific activity on a model substrate MeGlcA3Xyl4 and linear xylooligosaccharides.
• Molecular docking experiments to assess substrate binding modes and energies.

Main Results:

• Modifying the carboxyl group of GX substrates drastically reduced EcXyn30A's catalytic efficiency (several thousand-fold).
• The R293A mutation reduced catalytic efficiency on GX by only 18-fold, with similar kcat values across all tested polymers.
• Both enzymes cleaved substrates at the second glycosidic bond from the branch towards the reducing end; R293A showed reduced activity on MeGlcA3Xyl4 but not linear xylooligosaccharides.

Conclusions:

• Arg293 is essential for the specific recognition of the carboxyl group in 4-O-methylglucuronoxylan by EcXyn30A.
• The R293A mutation alters substrate specificity, suggesting Arg293's role in substrate binding energy and catalytic efficiency.
• Docking studies confirm analogous binding but different binding energies for modified substrates in both enzyme forms.