Linked Life Data

10600124

Source:http://linkedlifedata.com/resource/pubmed/id/10600124

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
50
pubmed:dateCreated
2000-1-19
pubmed:abstractText
The alpha(2)beta(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida is composed of a flavoprotein homodimer (alpha(2) or PchF(2); M(r) = 119 kDa) with a cytochrome monomer (beta, PchC; M(r) = 9.3 kDa) bound to each PchF subunit. Escherichia coli BL21(DE3) has been transformed with a vector for expression of the pchF gene, and PchF is overproduced by this strain as the homodimer. During purification, it was recognized that some PchF had FAD bound, while the remainder was FAD-free. However, unlike PchF obtained from PCMH purified from P. putida, FAD was bound noncovalently. The FAD was conveniently removed from purified E. coli-expressed PchF by hydroxyapatite chromatography. Fluorescence quenching titration indicated that the affinity of apo-PchF for FAD was sufficiently high to prevent the determination of the dissociation constant. It was found that p-cresol was virtually incapable of reducing PchF with noncovalently bound FAD (PchF(NC)), whereas 4-hydroxybenzyl alcohol, the intermediate product of p-cresol oxidation by PCMH, reduced PchF(NC) fairly quickly. In contrast, p-cresol rapidly reduced PchF with covalently bound FAD (PchF(C)), but, unlike intact PCMH, which consumed 4 electron equiv/mol when titrated with p-cresol (2 electrons from p-cresol and 2 from 4-hydroxybenzyl alcohol), PchF(C) accepted only 2 electron equiv/mol. This is explained by extremely slow release of 4-hydroxybenzyl alcohol from reduced PchF(C). 4-Hydroxybenzyl alcohol rapidly reduced PchF(C), producing 4-hydroxybenzaldehyde. It was demonstrated that p-cresol has a charge-transfer interaction with FAD when bound to oxidized PchF(NC), whereas 4-bromophenol (a substrate analogue) and 4-hydroxybenzaldehyde have charge-transfer interactions with FAD when bound to either PchF(C) or PchF(NC). This is the first example of a "wild-type" flavoprotein, which normally has covalently bound flavin, to bind flavin noncovalently in a stable, redox-active manner.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
http://linkedlifedata.com/resource/pubmed/chemical/4-cresol dehydrogenase..., http://linkedlifedata.com/resource/pubmed/chemical/4-hydroxybenzaldehyde, http://linkedlifedata.com/resource/pubmed/chemical/Bacterial Proteins, http://linkedlifedata.com/resource/pubmed/chemical/Benzaldehydes, http://linkedlifedata.com/resource/pubmed/chemical/Cytochromes, http://linkedlifedata.com/resource/pubmed/chemical/Dithionite, http://linkedlifedata.com/resource/pubmed/chemical/Flavin-Adenine Dinucleotide, http://linkedlifedata.com/resource/pubmed/chemical/Flavoproteins, http://linkedlifedata.com/resource/pubmed/chemical/Mixed Function Oxygenases, http://linkedlifedata.com/resource/pubmed/chemical/Peptide Synthases, http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Proteins, http://linkedlifedata.com/resource/pubmed/chemical/aryl-4,2-bisthiazoline synthetase...
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
14
pubmed:volume
38
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
16620-8
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Properties of p-cresol methylhydroxylase flavoprotein overproduced by Escherichia coli.
pubmed:affiliation
Molecular Biology Division, Department of Veterans Affairs Medical Center, San Francisco, California 94121, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S., Research Support, Non-U.S. Gov't