Linked Life Data

9179284

Source:http://linkedlifedata.com/resource/pubmed/id/9179284

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1997-7-14
pubmed:databankReference
pubmed:abstractText
Homoprotocatechuate (hpca, 3,4-dihydroxyphenylacetate) is a central intermediate for the bacterial degradation of aromatic compounds. Homoprotocatechuate 2,3-dioxygenase (HPCD) catalyzes the key ring cleavage step in the metabolism of hpca by the Gram (+) bacterium Brevibacterium fuscum to yield alpha-hydroxy-delta-carboxymethyl cis-muconic semialdehyde. A genomic DNA library of B. fuscum was constructed in Escherichia coli using a cosmid vector and screened by spraying the cells with hpca. One clone was found to contain the gene for HPCD based on its ability to convert hpca into the yellow-colored product. This cosmid clone was further subcloned and the gene for HPCD was localized and sequenced. The open reading frame codes for a protein with 365 amino acids and M(r) = 41,699, in accord with the characteristics of the previously purified wild-type enzyme. The gene for HPCD was overexpressed in E. coli to approximately 30% of the total soluble protein, and purification of the recombinant enzyme to apparent homogeneity was achieved by a two-step procedure. Iron was the only abundant metal found in the purified recombinant enzyme, and the specific activity per iron was comparable to that observed for the wild-type enzyme. The deduced amino acid sequence of HPCD has a very high level of homology (78.6% identity in the 337-aa overlap) to the manganese-dependent homoprotocatechuate 2,3-dioxygenase (MndD) from Arthrobacter globiformis CM-2. The basis for the difference in metal selection by HPCD and MndD was investigated by mutagenesis of a 50-base-pair region of the HPCD gene containing three frame shifts relative to the MndD gene. The purified triple mutant of HPCD did not exhibit a significant change in the metal content; therefore, other factors must contribute to the selection of the active site metal.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
1046-5928
pubmed:author
pubmed:issnType
Print
pubmed:volume
10
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1-9
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:9179284-3,4-Dihydroxyphenylacetic Acid, pubmed-meshheading:9179284-Amino Acid Sequence, pubmed-meshheading:9179284-Arthrobacter, pubmed-meshheading:9179284-Bacterial Proteins, pubmed-meshheading:9179284-Base Sequence, pubmed-meshheading:9179284-Biodegradation, Environmental, pubmed-meshheading:9179284-Brevibacterium, pubmed-meshheading:9179284-Cloning, Molecular, pubmed-meshheading:9179284-Cosmids, pubmed-meshheading:9179284-DNA, Bacterial, pubmed-meshheading:9179284-Dioxygenases, pubmed-meshheading:9179284-Escherichia coli, pubmed-meshheading:9179284-Frameshift Mutation, pubmed-meshheading:9179284-Gene Expression Regulation, Bacterial, pubmed-meshheading:9179284-Gene Library, pubmed-meshheading:9179284-Genes, Bacterial, pubmed-meshheading:9179284-Iron, pubmed-meshheading:9179284-Kinetics, pubmed-meshheading:9179284-Manganese, pubmed-meshheading:9179284-Molecular Sequence Data, pubmed-meshheading:9179284-Mutagenesis, Site-Directed, pubmed-meshheading:9179284-Open Reading Frames, pubmed-meshheading:9179284-Oxygenases, pubmed-meshheading:9179284-Recombinant Fusion Proteins, pubmed-meshheading:9179284-Sequence Alignment, pubmed-meshheading:9179284-Sequence Homology, Amino Acid, pubmed-meshheading:9179284-Species Specificity
pubmed:year
1997
pubmed:articleTitle
Cloning, overexpression, and mutagenesis of the gene for homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum.
pubmed:affiliation
Department of Biochemistry, Medical School, University of Minnesota, Minneapolis 55455, USA.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, U.S. Gov't, P.H.S.