Source:http://linkedlifedata.com/resource/pubmed/id/10559214
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rdf:type | |
lifeskim:mentions | |
pubmed:issue |
47
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pubmed:dateCreated |
1999-12-14
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pubmed:abstractText |
2-Hydroxybiphenyl 3-monooxygenase (EC 1.14.13.44) from Pseudomonas azelaica HBP1 is an FAD-dependent aromatic hydroxylase that catalyzes the conversion of 2-hydroxybiphenyl to 2, 3-dihydroxybiphenyl in the presence of NADH and oxygen. The catalytic mechanism of this three-substrate reaction was investigated at 7 degrees C by stopped-flow absorption spectroscopy. Various individual steps associated with catalysis were readily observed at pH 7.5, the optimum pH for enzyme turnover. Anaerobic reduction of the free enzyme by NADH is a biphasic process, most likely reflecting the presence of two distinct enzyme forms. Binding of 2-hydroxybiphenyl stimulated the rate of enzyme reduction by NADH by 2 orders of magnitude. The anaerobic reduction of the enzyme-substrate complex involved the formation of a transient charge-transfer complex between the reduced flavin and NAD(+). A similar transient intermediate was formed when the enzyme was complexed with the substrate analog 2-sec-butylphenol or with the non-substrate effector 2,3-dihydroxybiphenyl. Excess NAD(+) strongly stabilized the charge-transfer complexes but did not give rise to the appearance of any intermediate during the reduction of uncomplexed enzyme. Free reduced 2-hydroxybiphenyl 3-monooxygenase reacted rapidly with oxygen to form oxidized enzyme with no appearance of intermediates during this reaction. In the presence of 2-hydroxybiphenyl, two consecutive spectral intermediates were observed which were assigned to the flavin C(4a)-hydroperoxide and the flavin C(4a)-hydroxide, respectively. No oxygenated flavin intermediates were observed when the enzyme was in complex with 2, 3-dihydroxybiphenyl. Monovalent anions retarded the dehydration of the flavin C(4a)-hydroxide without stabilization of additional intermediates. The kinetic data for 2-hydroxybiphenyl 3-monooxygenase are consistent with a ternary complex mechanism in which the aromatic substrate has strict control in both the reductive and oxidative half-reaction in a way that reactions leading to substrate hydroxylation are favored over those leading to the futile formation of hydrogen peroxide. NAD(+) release from the reduced enzyme-substrate complex is the slowest step in catalysis.
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pubmed:language |
eng
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pubmed:journal | |
pubmed:citationSubset |
IM
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pubmed:chemical | |
pubmed:status |
MEDLINE
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pubmed:month |
Nov
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pubmed:issn |
0021-9258
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pubmed:author | |
pubmed:issnType |
Print
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pubmed:day |
19
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pubmed:volume |
274
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
33355-65
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pubmed:dateRevised |
2006-11-15
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pubmed:meshHeading |
pubmed-meshheading:10559214-Catalysis,
pubmed-meshheading:10559214-Kinetics,
pubmed-meshheading:10559214-Mixed Function Oxygenases,
pubmed-meshheading:10559214-NAD,
pubmed-meshheading:10559214-Protein Binding,
pubmed-meshheading:10559214-Pseudomonas,
pubmed-meshheading:10559214-Substrate Specificity
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pubmed:year |
1999
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pubmed:articleTitle |
Catalytic mechanism of 2-hydroxybiphenyl 3-monooxygenase, a flavoprotein from Pseudomonas azelaica HBP1.
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pubmed:affiliation |
Department of Microbiology, Swiss Federal Institute of Environmental Sciences and Technology (EAWAG), CH-8600 Dübendorf, The Netherlands.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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