The role of remote flavin adenine dinucleotide pieces in the oxidative decarboxylation catalyzed by salicylate hydroxylase

Abstract

Salicylate hydroxylase (NahG) has a single redox site in which FAD is reduced by NADH, the O₂ is activated by the reduced flavin, and salicylate undergoes an oxidative decarboxylation by a C(4a)-hydroperoxyflavin intermediate to give catechol. We report experimental results that show the contribution of individual pieces of the FAD cofactor to the observed enzymatic activity for turnover of the whole cofactor. A comparison of the kinetic parameters and products for the NahG-catalyzed reactions of FMN and riboflavin cofactor fragments reveal that the adenosine monophosphate (AMP) and ribitol phosphate pieces of FAD act to anchor the flavin to the enzyme and to direct the partitioning of the C(4a)-hydroperoxyflavin reaction intermediate towards hydroxylation of salicylate. The addition of AMP or ribitol phosphate pieces to solutions of the truncated flavins results in a partial restoration of the enzymatic activity lost upon truncation of FAD, and the pieces direct the reaction of the C(4a)-hydroperoxyflavin intermediate towards hydroxylation of salicylate.

Keywords: Activation; Biocatalysis; Flavoenzyme; One-component flavoprotein monooxygenase; Oxidoreductases.

Figure 1.

Kinetic profiles for (a) AMP and (b) ADP activation of NahG-catalyzed oxidation of NADH (200 μM) in the presence of salicylate (150 μM) and riboflavin (■), FMN (●), or FAD (▲), at pH 8.5, I = 0.21 M (NaCl), and 25 °C. The solid lines show the fits of the data to Eq. 1 using the kinetic parameters reported in Table 1.

Figure 2.

Kinetic profiles for exogenous (a) riboflavin, (b) FMN, and (c) FAD mediating the catalyzed oxidation of NADH (200 μM) by NahG in the presence of salicylate (150 μM) and in absence (■) and presence of AMP (○) and ADP (△) at pH 8.5, I = 0.21 M (NaCl), and 25 °C. Data fits were according to Eq. 1 and the kinetic parameters in Table 2.

Figure 3.

Homologs flavoproteins showing different conformational states at the FAD binding cleft: (a) “out” and (b) “in” states for FAD bound homologs showing the corresponding loop in PhzS from Pseudomonas aeruginosa (PDB entry 3C96) and 3HB6H from Rhodococcus jostii (PDB entry 4BJZ) in different positions, respectively.

Scheme 1.

Flavin structures and rationale for FAD truncation into different pieces.

Scheme 2.

Steps and mechanism along the pathways catalyzed by salicylate hydroxylase (NahG) using FAD as prosthetic group: 1. decarboxylative oxidation of salicylate (coupled path); 2. formation of hydrogen peroxide from a FAD-hydroperoxide species (uncoupled path).

Scheme 3.

Pathways for the unactivated and adenosyl nucleotide-activated NahG-catalyzed reactions of truncated flavins (Flv), where k_un and k_cp are apparent rate constants for reactions of the salicylate•hydroperoxyflavin complex to yield H₂O₂, and catechol and CO₂, respectively.