A comparative study of two α-L-rhamnosidases with high sequence identity

Area of Science:

• Enzymology
• Biochemistry
• Structural Biology

Background:

• The GH78 alpha-L-rhamnosidase from Aspergillus tubingensis (AT-Rha) represents a distinct group within Aspergillus enzymes.
• A highly similar enzyme, AK-Rha from A. kawachii IFO 4308, was investigated for comparative analysis.

Purpose of the Study:

• To express and characterize the recombinant AK-Rha (AK-rRha) from Aspergillus kawachii.
• To compare the enzymatic properties and structural features of AK-rRha with AT-Rha.
• To elucidate the structural basis for differences in catalytic efficiency between the two enzymes.

Main Methods:

• Recombinant protein expression in Pichia pastoris.
• Enzyme activity assays using naringin as a substrate.
• Molecular dynamics simulations to analyze enzyme conformation and flexibility.
• Polar solvation energy analysis to assess glycosidic bond hydrolysis.

Main Results:

• AK-rRha displayed significantly lower enzyme activity (0.816 U/mg) compared to AT-Rha (125.142 U/mg) towards naringin.
• The difference in catalytic efficiency was primarily attributed to a lower kcat value for AK-rRha (0.67 s⁻¹) versus AT-Rha (4.89 × 10⁴ s⁻¹).
• Molecular dynamics simulations revealed AK-Rha to be conformationally rigid, while AT-Rha was flexible, with Loop Y-L influencing naringin interaction and steric hindrance.

Conclusions:

• Both AK-Rha and AT-Rha exhibit narrow substrate specificity for naringin, characteristic of Aspergillus alpha-L-rhamnosidases.
• Structural flexibility and solvation energy significantly influence the catalytic efficiency of these enzymes.
• The study provides structural insights into the varying catalytic abilities of related enzymes.