Hydroquinone dioxygenase from pseudomonas fluorescens ACB: a novel member of the family of nonheme-iron(II)-dependent dioxygenases.
Hydroquinone 1,2-dioxygenase (HQDO), an enzyme involved in the catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB, was purified to apparent homogeneity. Ligandation with 4-hydroxybenzoate prevented the enzyme from irreversible inactivation. HQDO was activated by iron(II) ions and catalyzed the ring fission of a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes. HQDO was inactivated by 2,2'-dipyridyl, o-phenanthroline, and hydrogen peroxide and inhibited by phenolic compounds. The inhibition with 4-hydroxybenzoate (K(i) = 14 microM) was competitive with hydroquinone. Online size-exclusion chromatography-mass spectrometry revealed that HQDO is an alpha2beta2 heterotetramer of 112.4 kDa, which is composed of an alpha-subunit of 17.8 kDa and a beta-subunit of 38.3 kDa. Each beta-subunit binds one molecule of 4-hydroxybenzoate and one iron(II) ion. N-terminal sequencing and peptide mapping and sequencing based on matrix-assisted laser desorption ionization--two-stage time of flight analysis established that the HQDO subunits are encoded by neighboring open reading frames (hapC and hapD) of a gene cluster, implicated to be involved in 4-hydroxyacetophenone degradation. HQDO is a novel member of the family of nonheme-iron(II)-dependent dioxygenases. The enzyme shows insignificant sequence identity with known dioxygenases.
Moonen
Mariëlle J H
MJ
Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands. willem.vanberkel@wur.nl.
Synowsky
Silvia A
SA
van den Berg
Willy A M
WA
Westphal
Adrie H
AH
Heck
Albert J R
AJ
van den Heuvel
Robert H H
RH
Fraaije
Marco W
MW
van Berkel
Willem J H
WJ
D015082:2,2'-Dipyridyl / Q000494:pharmacology; D000098:Acetophenones / Q000378:metabolism; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D004269:DNA, Bacterial / Q000235:genetics; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006873:Hydroquinones / Q000378:metabolism*; D007501:Iron / Q000494:pharmacology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D005810:Multigene Family; D016366:Open Reading Frames; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010226:Parabens / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D021122:Protein Subunits / Q000737:chemistry; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D000098:Acetophenones; D004269:DNA, Bacterial; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D005231:Fatty Acids, Unsaturated; D006873:Hydroquinones; D010226:Parabens; D010618:Phenanthrolines; D021122:Protein Subunits; C002091:hydroxymuconic semialdehyde; D015082:2,2'-Dipyridyl; D006861:Hydrogen Peroxide; D007501:Iron; D010105:Oxygenases; C400568:(chloro)hydroquinone 1, 2-dioxygenase; C031335:4-hydroxyacetophenone; C038193:4-hydroxybenzoic acid; C025205:1,10-phenanthroline; C031927:hydroquinone