Purification and characteriation of 3‐hydroxyphenylacetate 6‐hydroxylase: a novel FAD‐dependent monooxygenase from a Flavobacterium species

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Abstract
3‐Hydroxyphenylacetate 6‐hyroxylase was purified 70‐fold from a Flavobacterium sp. grown upon phenylactic acid as its sole carbon and energy source. The presence of FAD and dithiothreitol during purification is essential for high recovery of active enzyme.SDS/PAGE of purified enzyme reveals a single band with a minimum molecuarl mass of 63 KDa. Analytical gel‐filtration, sedimentation‐equilibrium and sedimentation‐velocity experiments indicate that the purified enzyme exists in solultion manily as a dimer, containing 1 molecule non‐covalently bound FAD/sununit.3‐Hydroxyphenylacetate 6‐hydroxylase utilizes NADH and NADPH as external electron donors with similar efficiency. The enzyme shows a narrow substrate specificity. Only the primary substrate 3‐hydroxyphenylacetate is hydroxylated efficiently, yielding 2.5‐dihydroxyphenylacetate as a product. During turnover, the substrate analogues 3,4‐dihydroxyphenylacetate and 4‐hydroxyphenylacetate are partially hydroxylated, exculsively at the 6′ (2′) positition. The physiological product 2.5‐dihydroxyphenylacetate acts as an effector, strongly stimulating NAD(P)H oxidation. The activity of 3‐hydroxyphenylacetate 6‐hydroxylase is severely inhibited by chloride ions, competitive to the aromatic substrate.In the native state of enzyme, two sulfhydryl groups are accessible to 5.5′‐dithiobis(2‐nitrobenzoate). Titration with Stoichiometric amounts of either 5.5′‐dithiobis(2‐nitrobenzoate) or mercurial reagents completely blocks enzyme activity. Inactivation by cysteine reagents is inhibited by the substrate 3‐hydroxyphenylacetate. The original activity is fully restored by treatment of the modified enzyme with dithiothreitol.The N‐terminal amino acid sequence of the enzyme lacks the consenusus sequence GXGXXG, found at the termini of all faavin‐dependent external mnooxygenses sequenced so far. The amino acid composition of 3‐hydroxyphenylacetate 6‐hydroxylase is also presented.

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