Functional Analysis of the Small Component of the 4-Hydroxyphenylacetate 3-Monooxygenase ofEscherichia coliW: a Prototype of a New Flavin:NAD(P)H Reductase Subfamily

• DOI: 10.1128/jb.182.3.627-636.2000
• Publication Date: 2002-07-27T10:01:09Z
• Authors: Beatriz Galán; Eduardo Díaz; María A. Prieto; José L. García
• Keywords: 0301 basic medicine; Base Sequence; FMN Reductase; Molecular Sequence Data; Restriction Mapping; Mixed Function Oxygenases; 3. Good health; Evolution, Molecular; Structure-Activity Relationship; 03 medical and health sciences; Escherichia coli; NADH, NADPH Oxidoreductases; Amino Acid Sequence; Sequence Alignment

ABSTRACTEscherichia coliW uses the aromatic compound 4-hydroxyphenylacetate (4-HPA) as a sole source of carbon and energy for growth. The monooxygenase which converts 4-HPA into 3,4-dihydroxyphenylacetate, the first intermediate of the pathway, consists of two components, HpaB (58.7 kDa) and HpaC (18.6 kDa), encoded by thehpaBandhpaCgenes, respectively, that form a single transcription unit. Overproduction of the small HpaC component inE. coliK-12 cells has facilitated the purification of the protein, which was revealed to be a homodimer that catalyzes the reduction of free flavins by NADH in preference to NADPH. Subsequently, the reduced flavins diffuse to the large HpaB component or to other electron acceptors such as cytochromecand ferric ion. Amino acid sequence comparisons revealed that the HpaC reductase could be considered the prototype of a new subfamily of flavin:NAD(P)H reductases. The construction of a fusion protein between the large HpaB oxygenase component and the choline-binding domain of the major autolysin ofStreptococcus pneumoniaeallowed us to develop a rapid method to efficiently purify this highly unstable enzyme as a chimeric CH-HpaB protein, which exhibited a 4-HPA hydroxylating activity only when it was supplemented with the HpaC reductase. These results suggest the 4-HPA 3-monooxygenase ofE. coliW as a representative member of a novel two-component flavin-diffusible monooxygenase (TC-FDM) family. Relevant features on the evolution and structure-function relationships of these TC-FDM proteins are discussed.