Linked Life Data

9344474

Source:http://linkedlifedata.com/resource/pubmed/id/9344474

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1997-12-2
pubmed:abstractText
Recombinant adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca overexpressed in Escherichia coli was purified to homogeneity. The enzyme has a low solubility and was extracted from the crude membrane fraction with 1% Brij 35 in a high recovery. Subsequent chromatography on DEAE-cellulose resulted in 4.9-fold purification of the enzyme in an overall yield of 65%. The enzyme thus obtained showed specific activity comparable to that of the wild-type enzyme of K. oxytoca. The apparent molecular weight determined by nondenaturing gel electrophoresis on a gradient gel was 220,000. The enzyme consists of equimolar amounts of the three subunits with apparent Mr of 60,000 (alpha), 30,000 (beta), and 19,000 (gamma). Therefore, the subunit structure of the enzyme is most likely alpha2beta2gamma2. The recombinant enzyme was also separated into components F and S upon DEAE-cellulose chromatography in the absence of substrate. Components F and S were identified as the beta subunit and alpha2gamma2 complex, respectively. Apparent Km for adenosylcobalamin, 1,2-propanediol, glycerol, and 1,2-ethanediol were 0.83 microM, 0.08 mM, 0.73 mM, and 0.56 mM, respectively. The three genes encoding the subunits of diol dehydratase were overexpressed individually or in various combinations in Escherichia coli. The alpha and gamma subunits mutually required each other for correct folding forming the soluble, active alpha2gamma2 complex (component S). Expression of the beta subunit in a soluble, active form (component F) was promoted by coexpression with both the alpha and gamma subunits, probably by coexistence with component S. These lines of evidence indicate that each subunit mutually affects the folding of the others in this heterooligomer enzyme.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
0003-9861
pubmed:author
pubmed:copyrightInfo
Copyright 1997 Academic Press.
pubmed:issnType
Print
pubmed:day
1
pubmed:volume
347
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
132-40
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:9344474-Chromatography, Ion Exchange, pubmed-meshheading:9344474-Cobamides, pubmed-meshheading:9344474-Detergents, pubmed-meshheading:9344474-Electrophoresis, Gel, Two-Dimensional, pubmed-meshheading:9344474-Electrophoresis, Polyacrylamide Gel, pubmed-meshheading:9344474-Escherichia coli, pubmed-meshheading:9344474-Gene Expression, pubmed-meshheading:9344474-Genes, Bacterial, pubmed-meshheading:9344474-Kinetics, pubmed-meshheading:9344474-Klebsiella, pubmed-meshheading:9344474-Molecular Weight, pubmed-meshheading:9344474-Peptide Fragments, pubmed-meshheading:9344474-Polyethylene Glycols, pubmed-meshheading:9344474-Propanediol Dehydratase, pubmed-meshheading:9344474-Protein Conformation, pubmed-meshheading:9344474-Protein Folding, pubmed-meshheading:9344474-Recombinant Proteins, pubmed-meshheading:9344474-Sequence Analysis
pubmed:year
1997
pubmed:articleTitle
Heterologous expression, purification, and properties of diol dehydratase, an adenosylcobalamin-dependent enzyme of Klebsiella oxytoca.
pubmed:affiliation
Faculty of Engineering, Okayama University, Okayama, Tsushima-Naka, 700, Japan.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't