Coordinated production and utilization of FADH2 by NAD(P)H-flavin oxidoreductase and 4-hydroxyphenylacetate 3-monooxygenase.
4-Hydroxyphenylacetate (4HPA) 3-monooxygenase (HpaB) is a reduced flavin adenine dinucleotide (FADH(2)) utilizing monooxygenase. Its cosubstrate, FADH(2), is supplied by HpaC, an NAD(P)H-flavin oxidoreductase. Because HpaB is the first enzyme for 4HPA metabolism, FADH(2) production and utilization become a major metabolic event when Escherichia coli W grows on 4HPA. An important question is how FADH(2) is produced and used, as FADH(2) is unstable in the presence of free O(2). One solution is metabolic channeling by forming a transitory HpaB-HpaC complex. However, our in vivo and in vitro data failed to support the interaction. Further investigation pointed to an alternative scheme for HpaB to sequester FADH(2). The intracellular HpaB concentration was about 122 microM in 4HPA-growing cells, much higher than the total intracellular FAD concentration, and HpaB had a high affinity for FADH(2) (K(d) of 70 nM), suggesting that most FADH(2) is bound to HpaB in vivo. The HpaB-bound FADH(2) was either used to rapidly oxidize 4HPA or slowly oxidized by O(2) to FAD and H(2)O(2) in the absence of 4HPA. Thus, HpaB's high intracellular concentration, its high affinity for FADH(2), its property of protecting bound FADH(2) in the absence of 4HPA, and its ability to rapidly use FADH(2) to oxidize 4HPA when 4HPA is available can coordinate FADH(2) production and utilization by HpaB and HpaC in vivo. This type of coordination, in responding to demand, for production and utilization of labile metabolites has not been reported to date.
Louie
Tai Man
TM
School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4234, USA.
Xie
X Sunney
XS
Xun
Luying
L
D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D038181:FMN Reductase / Q000235:genetics / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009154:Mutation; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010648:Phenylacetates / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D013050:Spectrometry, Fluorescence / Q000379:methods
D017931:DNA Primers; D010648:Phenylacetates; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; C026246:4-hydroxyphenylacetate; D006899:Mixed Function Oxygenases; C026252:4-hydroxyphenylacetate 3-monooxygenase; D038181:FMN Reductase