Identification of key amino acid residues governing substrate-entry, catalysis and dimerization in 1-naphthol-2-hydroxylase from Pseudomonas sp. C5pp.
TL;DR
The C-terminal domain was crucial for folding and dimerization, while H541A/Y542A led to monomeric 1NH with ∼52% activity, providing insights into role of domains and the catalytic mechanism of 1NH.
Abstract
1-Naphthol-2-hydroxylase (1NH, group-A flavoprotein monooxygenase) from Carbaryl-degrading Pseudomonas sp. C5pp catalyzes ortho-hydroxylation of 1-naphthol to 1,2-dihydroxynaphthalene. Homology model-guided site-directed-mutagenesis identified residues involved in FAD-binding, substrate-entry, catalysis and dimerization. Alanine substitution of R55, Q128, W298 and D318 caused activity loss with decreased flavin content, indicating roles in FAD-binding. Bulky substitutions in tunnel lumen (G207F, M260F, M380F and M383F) abolished activity, supporting its role in substrate-entry. Mutations at the active-site showed that H57 forms principal substrate-binding residue which is oriented by N125, while L229 and P325 contribute to substrate positioning. The apparent coupling efficiency (hydroxylation efficiency) of mutants remained comparable to that of wildtype enzyme, indicating that these substitutions affected activity without altering coupling between NADH oxidation and product formation. The C-terminal domain was crucial for folding and dimerization, while H541A/Y542A led to monomeric 1NH with ∼52% activity. These findings provide insights into role of domains and the catalytic mechanism of 1NH.