Enzymes melilotate hydroxylase

Infrared spectroscopy was one of the tools used (Levy and Frost, 1966) for a definitive identification of 2,3-dihydroxyphenylpropionic add, the product from the reaction between the enzyme, melilotate hydroxylase (from extracts of the Arthrobac-ter species), and melilotic acid, NADH, and oxygen. 

The mechanism for the hydroxylation of aromatic substrates by flavoprotein monooxygenases has been the subject of signiflcant research interest and controversy over the past decade. These enzymes (p-hydroxybenzoate hydroxylase, phenol hydroxylase, and melilotate hydroxylase) catalyze the initial step in the )8-ketoadipic acid pathway, the hydroxylation of substituted phenols into catechols (Scheme 55). Oxygen is required as cosubstrate, which is activated by the reduced FAD cofactor. The complex mechanism for the oxidative half-reaction is thought to consist of at least four steps and three intermediates 239-242) and to involve a controversial 4a,5-ring-opened flavin 242, 249, 250) (Scheme 56). The flavin C4a-hydroperoxy intermediate 64 and flavin C4a-hydroxy intermediate 65 have been assigned the structures shown in Scheme 56 based on the UV absorbance spectra of various model compounds compared with that of the modified enzyme cofactor alkylated at N(5) 243). However, evidence for the intermediacy of various ring-opened flavin species has been tentative at best, as model compounds and model reactions do not support such an intermediate 242). 

This FAD-dependent enzyme [EC 1.14.13.4], also known as melilotate 3-monooxygenase and 2-hydroxyphenyl-propionate hydroxylase, catalyzes the reaction of 3-(2-hydroxyphenyl)propanoate with NADH and dioxygen to produce 3-(2,3-dihydroxyphenyl)propanoate, NAD+, and water. 

---