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Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase
Jian Wang1, Mariliz Ortiz-Maldonado, Barrie Entsch
1Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Summary
Para-hydroxybenzoate hydroxylase uses dynamic protein movements to control solvent access for its two-step reaction. A mutant enzyme reveals how flavin cofactor positioning and substrate entry facilitate catalysis.
Area of Science:
- Biochemistry
- Enzyme catalysis
- Protein dynamics
Background:
- Para-hydroxybenzoate hydroxylase (PHBH) catalyzes a crucial two-step reaction.
- Precise control of solvent access to the catalytic site is essential for PHBH activity.
- Conflicting solvent requirements for flavin reduction and oxygenation necessitate complex protein dynamics.
Purpose of the Study:
- To investigate the role of protein dynamics in PHBH catalysis using a specific mutant.
- To elucidate the mechanism of substrate entry and cofactor binding in PHBH.
- To understand how PHBH coordinates solvent access for its two distinct reaction steps.
Main Methods:
- Structural analysis of the R220Q mutant of para-hydroxybenzoate hydroxylase.
- Determination of the complex structure with pyridine nucleotide.
- Analysis of protein domain movements and cofactor positioning.
Main Results:
- In the R220Q mutant, domain separation exposes the flavin cofactor to solvent in the absence of substrate.
- Substrate enters the active site via a surface binding site followed by sliding into the interior.
- Pyridine nucleotide (NADPH) binds in an extended conformation, suggesting a mechanism for stereospecific reduction.
Conclusions:
- Complex protein dynamics, including domain movements and cofactor repositioning, are central to PHBH catalysis.
- The enzyme dynamically regulates solvent access to accommodate different reaction steps.
- Understanding these dynamics provides insights into enzyme mechanism and engineering potential.