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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
29
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pubmed:dateCreated |
1992-11-18
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pubmed:databankReference |
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pubmed:abstractText |
The amino acid sequences of the NH2 terminus and internal peptide fragments of a Rhodococcus rhodochrous J1 nitrilase were determined to prepare synthetic oligonucleotides as primers for the polymerase chain reaction. A 750-base DNA fragment thus amplified was used as the probe to clone a 5.4-kilobase PstI fragment coding for the whole nitrilase. The nitrilase gene modified in the sequence upstream from the presumed ATG start codon was expressed to approximately 50% of the total soluble protein in Escherichia coli. The predicted amino acid sequence of the nitrilase gene showed similarity to that of the bromoxynil nitrilase from Klebsiella ozaenae. The 5,5'-dithiobis(2-nitrobenzoic acid) modification of the nitrilase from R. rhodochrous J1 resulted in inactivation with the loss of one sulfhydryl group/enzyme subunit. Of 4 cysteine residues in the Rhodococcus nitrilase, only Cys-165 is conserved in the Klebsiella nitrilase. Mutant enzymes containing Ala or Ser instead of Cys-165 did not exhibit nitrilase activity. These findings suggest that Cys-165 plays an essential role in the function of the active site.
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
Oct
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pubmed:issn |
0021-9258
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:day |
15
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pubmed:volume |
267
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
20746-51
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pubmed:dateRevised |
2006-11-15
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pubmed:meshHeading |
pubmed-meshheading:1400390-Amino Acid Sequence,
pubmed-meshheading:1400390-Aminohydrolases,
pubmed-meshheading:1400390-Base Sequence,
pubmed-meshheading:1400390-Cloning, Molecular,
pubmed-meshheading:1400390-Cysteine,
pubmed-meshheading:1400390-DNA, Bacterial,
pubmed-meshheading:1400390-Electrophoresis, Polyacrylamide Gel,
pubmed-meshheading:1400390-Escherichia coli,
pubmed-meshheading:1400390-Genes, Bacterial,
pubmed-meshheading:1400390-Kinetics,
pubmed-meshheading:1400390-Molecular Sequence Data,
pubmed-meshheading:1400390-Oligodeoxyribonucleotides,
pubmed-meshheading:1400390-Polymerase Chain Reaction,
pubmed-meshheading:1400390-Recombinant Proteins,
pubmed-meshheading:1400390-Restriction Mapping,
pubmed-meshheading:1400390-Rhodococcus,
pubmed-meshheading:1400390-Sequence Homology, Amino Acid
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pubmed:year |
1992
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pubmed:articleTitle |
Nitrilase from Rhodococcus rhodochrous J1. Sequencing and overexpression of the gene and identification of an essential cysteine residue.
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pubmed:affiliation |
Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Japan.
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pubmed:publicationType |
Journal Article,
Comparative Study,
Research Support, Non-U.S. Gov't
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