Linked Life Data

2180940

Source:http://linkedlifedata.com/resource/pubmed/id/2180940

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
11
pubmed:dateCreated
1990-5-10
pubmed:databankReference
pubmed:abstractText
The oxidation of camphor by cytochrome P-450cam requires the participation of a flavoprotein, putidaredoxin reductase, and an iron-sulfur protein, putidaredoxin, to mediate the transfer of electrons from NADH to P-450 for oxygen activation. A 2.2-kilobase pair BamHI-StuI fragment from whole cell DNA of camphor-grown Pseudomonas putida has been cloned and sequenced. Translation of the sequence revealed two open reading frames that could code for putidaredoxin reductase and putidaredoxin. In the case of putidaredoxin, the translated sequence matched the published sequence (Tanaka, M., Haniu, M., Yasunobu, K. T., Dus, K., and Gunsalus, I. C. (1974) J. Biol. Chem. 249, 3689-3701) with the exception of one amino acid. Codon usage in these proteins, like the proteins of other Pseudomonads, is strongly biased to G + C in the third nucleotide. A potential transcription termination site was found 3' to the putidaredoxin coding region. The "FAD-binding" amino acid consensus sequence, present in other flavoproteins, was found in putidaredoxin reductase beginning at residue 11 and a second occurrence of this sequence was found beginning with amino acid 156. The second sequence could represent the NAD-binding site. The regions encoding putidaredoxin reductase and putidaredoxin were subcloned and independently expressed in Escherichia coli at the level of 0.4 and 4.8 mg of enzymatically active protein/g wet weight of cells, respectively. Site-directed mutagenesis was used to change the rare start codon, GTG, of putidaredoxin reductase to ATG which resulted in an 18-fold increase in the level of expression of this protein to 7.4 mg/g wet weight of cells. The construction of these two clones, which express these important proteins, will facilitate studies of their interaction with each other and with P-450cam.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
265
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
6066-73
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:2180940-Amino Acid Sequence, pubmed-meshheading:2180940-Base Sequence, pubmed-meshheading:2180940-Blotting, Southern, pubmed-meshheading:2180940-Cloning, Molecular, pubmed-meshheading:2180940-Codon, pubmed-meshheading:2180940-DNA, Bacterial, pubmed-meshheading:2180940-Escherichia coli, pubmed-meshheading:2180940-Ferredoxins, pubmed-meshheading:2180940-Gene Library, pubmed-meshheading:2180940-Molecular Sequence Data, pubmed-meshheading:2180940-Mutation, pubmed-meshheading:2180940-NADH, NADPH Oxidoreductases, pubmed-meshheading:2180940-Nucleic Acid Hybridization, pubmed-meshheading:2180940-Oligonucleotide Probes, pubmed-meshheading:2180940-Plasmids, pubmed-meshheading:2180940-Pseudomonas, pubmed-meshheading:2180940-Recombinant Proteins, pubmed-meshheading:2180940-Restriction Mapping, pubmed-meshheading:2180940-Sequence Homology, Nucleic Acid
pubmed:year
1990
pubmed:articleTitle
Putidaredoxin reductase and putidaredoxin. Cloning, sequence determination, and heterologous expression of the proteins.
pubmed:affiliation
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9038.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't