Linked Life Data

17823232

Source:http://linkedlifedata.com/resource/pubmed/id/17823232

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
12
pubmed:dateCreated
2007-11-21
pubmed:abstractText
The cytochrome P450 enzymes (P450s) that mediate mammalian xenobiotic metabolism are highly versatile monooxygenases, which show wide and overlapping substrate ranges but generally poor catalytic rates. Re-engineering of these P450s may enable the development of useful biocatalysts for industrial applications. In the current study, restriction enzyme-mediated DNA family shuffling was used to create a library from human CYP1A1 and CYP1A2. Among sequenced clones (four randomly selected and eight functional clones), 5.9 +/- 2.3 crossovers and 1.5 +/- 1.5 spontaneous mutations (mean +/- S.D.) were detected per mutant. A high level of structural integrity as well as diverse functionality were found, with 53% of clones expressed at significant levels (>50 nM P450 hemoprotein) and 23% of clones showing activity on one or more of the following compounds: luciferin 6'-chloroethyl ether (luciferin-CEE), luciferin 6'-methyl ether (luciferin-ME), 6'-deoxyluciferin (luciferin-H), the ethylene glycol ester of luciferin 6'-methyl ether, 7-ethoxyresorufin, and p-nitrophenol (PNP). Different activity profiles were seen with higher specific activity on individual compounds (e.g., clone 22; 9 times the CYP1A1 specific activity toward luciferin-CEE), novel activities (e.g., clone 35; activity toward luciferin-H and PNP), and broadening of substrate range observed in particular clones (e.g., clone 9; activity toward both selective substrates luciferin-ME and luciferin-CEE as well as toward luciferin-H and PNP). In summary, forms were found with distinct and novel activity profiles, despite the relatively small number of mutants examined. In addition, the whole-cell metabolic assays described here provide simple, high-throughput methods useful for screening larger libraries.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0090-9556
pubmed:author
pubmed:issnType
Print
pubmed:volume
35
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
2177-85
pubmed:meshHeading
pubmed:year
2007
pubmed:articleTitle
A shuffled CYP1A library shows both structural integrity and functional diversity.
pubmed:affiliation
Physiology and Pharmacology, School of Biomedical Sciences, The University of Queensland, St Lucia, Brisbane, Australia.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't, Evaluation Studies