Molecular modeling reveals the possible importance of a carbonyl oxygen binding pocket for the catalytic mechanism of p-hydroxybenzoate hydroxylase.
p-Hydroxybenzoate hydroxylase catalyzes the hydroxylation of an aromatic substrate and uses flavin as a cofactor. The reaction probably occurs via a flavin 4a-hydroperoxide intermediate. In this study the crystal structure of 4a,5-epoxyethano-3-methyl-4a,5-dihydrolumiflavin, an analogue of the flavin 4a-hydroperoxide intermediate, was fitted to the active site in the crystal structure of the p-hydroxybenzoate hydroxylase-3,4-dihydroxybenzoate complex. This model of an important catalytic intermediate fitted very well in the active site of p-hydroxybenzoate hydroxylase. The most striking result was that whereas with the normal flavin, the 0-4 of the flavin ring makes only poor hydrogen bonds with the protein, with the flavin 4a-hydroperoxide analogue, the same 0-4 makes strong hydrogen bonds with the NH groups of Gly-46 and Val-47. These two NH groups form a carbonyl oxygen binding pocket which has a geometry almost identical to the oxyanion hole found in several proteases. The possible consequences of this model for the reaction mechanism of p-hydroxybenzoate hydroxylase are discussed.
Schreuder
H A
HA
Laboratory of Chemical Physics, Groningen, The Netherlands.
Hol
W G
WG
Drenth
J
J
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000378:metabolism*; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D003460:Crystallization; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism; D006860:Hydrogen Bonding; D006900:Hydroxylation; D009682:Magnetic Resonance Spectroscopy; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008968:Molecular Conformation; D009249:NADP / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*
D005415:Flavins; C055063:4a,5-epoxyethano-3-methyl-4a,5-dihydrolumiflavin; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D006899:Mixed Function Oxygenases; D015109:4-Hydroxybenzoate-3-Monooxygenase; D010100:Oxygen