Structure, dimeric conformation, and coenzyme versatility of p-hydroxybenzoate hydroxylase from Arthrobacter sp....

Area of Science:

• Biochemistry
• Enzymology
• Protein Structure and Function

Background:

• p-Hydroxybenzoate hydroxylase (PHBH) enzymes catalyze the ortho-hydroxylation of 4-hydroxybenzoate (4-HB) to protocatechuate (PCA).
• PHBHs are typically homodimers, and understanding their pyridine nucleotide binding and specificity is crucial for biotechnological applications.
• The dimeric interface and coenzyme binding determinants in AspPHBH from Arthrobacter sp. PAMC25564 require further elucidation.

Purpose of the Study:

• To determine the dimerization interface of AspPHBH.
• To identify key residues involved in pyridine nucleotide binding and coenzyme specificity.
• To investigate the impact of mutations on coenzyme binding and enzymatic activity.

Main Methods:

• Crystallography to analyze the quaternary structure.
• Site-directed mutagenesis to probe residue function.
• Enzyme activity assays to assess coenzyme utilization and specificity.

Main Results:

• AspPHBH forms a functional dimer, consistent with previous studies, despite appearing as a tetramer in crystallographic asymmetric units.
• AspPHBH demonstrates coenzyme versatility, binding both NADH and NADPH, with a preference for NADH.
• Mutations in coenzyme-surrounding residues, particularly R50, R273, S166, and E44, significantly affected NADPH binding and specificity, impacting overall enzymatic activity.

Conclusions:

• The study elucidates the determinants of PHBH's active dimeric conformation and coenzyme binding specificity.
• Key residues R50, R273, S166, and E44 are critical for NAD(P)H binding and coenzyme specificity in AspPHBH.
• These findings provide a foundation for future biotechnological advancements involving PHBH enzymes.