Histidine 90 function in 4-chlorobenzoyl-coenzyme a dehalogenase catalysis

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• 4-chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase facilitates hydrolytic dehalogenation.
• The catalytic mechanism involves a Meisenheimer intermediate (EMc) and an arylated enzyme intermediate (EAr).
• The role of active site His90 in this pathway requires elucidation.

Purpose of the Study:

• To investigate the catalytic contribution of His90 in 4-CBA-CoA dehalogenase.
• To characterize the impact of His90 substitution on the enzyme's reaction pathway and kinetics.

Main Methods:

• Site-directed mutagenesis was employed to replace His90 with glutamine (H90Q mutant).
• X-ray crystallography was used to determine the structure of the H90Q mutant complexed with 4-hydroxybenzoyl-CoA (4-HBA-CoA).
• Kinetic techniques were applied to assess ligand binding and catalytic turnover rates for both wild-type and mutant enzymes.

Main Results:

• X-ray crystallography revealed no significant changes in catalytic group positions in the H90Q mutant, except for the Gln90 side chain rotation and water molecule occupation.
• Ligand binding rates for 4-CBA-CoA and 4-HBA-CoA were similar between wild-type and H90Q dehalogenases.
• The H90Q mutation drastically reduced catalytic turnover rates, with a 133-fold decrease in EAr formation and a 154-fold decrease in EAr hydrolysis rates.

Conclusions:

• His90 is essential for efficient catalysis, likely acting as a general base catalyst for ester hydrolysis.
• Active site hydration and a potential slow conformational change may contribute to the reduced catalytic efficiency in the H90Q mutant.
• The findings highlight the critical role of His90 in the dehalogenation mechanism of 4-CBA-CoA dehalogenase.