Structural Comparison of Substrate Binding Sites in Dehaloperoxidase A and B

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Dehaloperoxidase (DHP) exhibits diverse catalytic activities influenced by substrate binding, pH, and heme pocket dynamics.
• Enzyme kinetics indicate DHP B is significantly more catalytically active than DHP A, despite 96% sequence homology.
• Over 30 substrate-bound structures exist for DHP B, offering insights into active site binding, while DHP A structures are limited to phenols.

Purpose of the Study:

• To investigate substrate binding in DHP A and compare it with known DHP B structures.
• To test if substrates binding to DHP B can also bind to DHP A.
• To elucidate the relationship between substrate structure, binding orientation, and DHP enzymatic function.

Main Methods:

• Soaking of fifteen selected substrates, known to bind DHP B, into DHP A crystals.
• X-ray crystallography to determine the structures of substrate-bound DHP A.
• Analysis of electron density maps to confirm substrate presence and orientation.

Main Results:

• Five substrates (2,4-dichlorophenol, 2,4-dibromophenol, 4-nitrophenol, 4-nitrocatechol, 4-bromo-o-cresol) yielded high-quality X-ray crystal structures with DHP A.
• Larger substrates, including cresols, 5-bromoindole, benzimidazole, and biphenols, did not yield sufficient electron density in DHP A for structural determination.
• Direct structural comparisons of substrate binding modes in DHP A and DHP B were achieved for five substrates.

Conclusions:

• DHP A exhibits a more restricted substrate-binding profile compared to DHP B, with smaller phenolic compounds binding effectively.
• The binding conformation and orientation of substrates in DHP A are critical determinants of its enzymatic function.
• Findings support the hypothesis that substrate-binding conformation dictates the enzymatic activity of dehaloperoxidase.