Metabolic function and properties of 4-hydroxyphenylacetic acid 1-hydroxylase from Pseudomonas acidovorans

J Bacteriol. 1975 Jan;121(1):272-85. doi: 10.1128/jb.121.1.272-285.1975.


The enzyme 4-hydroxyphenylacetate, NAD(P)H:oxygen oxidoreductase (1-hydroxylating) (EC 1.14.13 ...; 4-hydroxyphenylacetate 1-monooxygenase; referred to here as 4-HPA 1-hydroxylase) was induced in Pseudomonas acidovorans when 4-hydroxyphenylacetate (4-PHA) was utilized as carbon source for growth; homogentisate and maleylacetoacetate were intermediates in the degradation of 4-HPA. A preparation of the hydroxylase that was free from homogentisate dioxygenase and could be stored at 4 C in the presence of dithioerythritol with little loss of activity was obtained by ultracentrifuging cell extracts; but when purified 18-fold by affinity chromatography the enzyme became unstable. Flavin adenine dinucleotide and Mg2+ ions were required for full activity. 4-HPA 1-hydrocylase was inhibited by KCl, which was uncompetitive with 4-HPA. Values of Ki determined for inhibitors competitive with 4-HPA were 17 muM dl-4-hydroxymandelic acid, 43 muM 3,4-dihydroxyphenylacetic acid, 87 muM 4-hydroxy-3-methylphenylacetic acid, and 440 muM 4-hydroxyphenylpropionic acid. Apparent Km values for substrates of 4-HPA 1-hydroxylase were 31 muM 4-HPA, 67 muM oxygen, 95 muM reduced nicotinamide adenine dinucleotide (NADH); AND 250 muM reduced nicotinamide adenine dinucleotide phosphate (NADPH). The same maximum velocity was given by NADH and NADPH. A chemical synthesis is described for 2-deutero-4-hydroxyphenylacetic acid. This compound was enzymatically hydroxylated with retention of half the deuterium in the homogentisic acid formed. Activity as substrate or inhibitor of 4-HPA 1-hydroxylase was shown only by those analogues of 4-HPA that possessed a hydroxyl group substituent at C-4 of the benze nucleus. A mechanism is suggested that accounts for this structural requirement and also for the observation that when 4-hydroxyphenoxyacetic acid was attacked by the enzyme, hydroquinone was formed by release of the side chain, probably as glycolic acid. Only one enantiometer of racemic 4-hydroxyhydratropic acid was attacked by 4-HPA 1-hydroxylase; the product, alpha-methylhomogentisic acid (2-(2,5-dihydroxyphenyl)-propionic acid), exhibited optical activity. This observation suggests that, during its shift from C-1 to C-2 of the nucleus, the side chain of the substrate remains bound to a site on the enzyme while a conformational change of the protein permits the necessary movement of the benzene ring.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell-Free System
  • Chemical Phenomena
  • Chemistry
  • Chromatography, Affinity
  • Chromatography, DEAE-Cellulose
  • Dithioerythritol
  • Enzyme Induction
  • Glutathione / metabolism
  • Homogentisic Acid / metabolism
  • Kinetics
  • Magnesium / metabolism
  • Mixed Function Oxygenases / antagonists & inhibitors
  • Mixed Function Oxygenases / metabolism*
  • NAD / metabolism
  • NADP / metabolism
  • Oxidation-Reduction
  • Oxygen Consumption
  • Phenanthrolines / metabolism
  • Phenylacetates / pharmacology
  • Potassium Chloride / pharmacology
  • Pseudomonas / enzymology*
  • Pseudomonas / metabolism
  • Ultracentrifugation


  • Phenanthrolines
  • Phenylacetates
  • NAD
  • NADP
  • Potassium Chloride
  • Dithioerythritol
  • Mixed Function Oxygenases
  • Glutathione
  • Magnesium
  • Homogentisic Acid