Functional analysis of the small component of the 4-hydroxyphenylacetate
3-monooxygenase of Escherichia coli W: a prototype of a new Flavin:NAD(P)H
reductase subfamily.
J Bacteriol, 182 (3):
627--636 (February 2000)Abstract
Escherichia coli W 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 the hpaB and hpaC genes, respectively,
that form a single transcription unit. Overproduction of the small
HpaC component in E. coli K-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 cytochrome c and 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
of Streptococcus pneumoniae allowed 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 of E. coli W 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.