Linked Life Data

1909895

Source:http://linkedlifedata.com/resource/pubmed/id/1909895

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
39
pubmed:dateCreated
1991-10-24
pubmed:abstractText
The cytoplasmic C-terminal domain, residues 348-637, and the membrane-bound N-terminal domain, residues 1-347, of EIImtl have been subcloned and expressed in Escherichia coli. The N-terminal domain, IICmtl, contains the mannitol binding site, and the C-terminal domain, IIBAmtl, contains the activity-linked phosphorylation sites, His-554 and Cys-384. Overexpression of the BA domain was achieved by a translational in-frame fusion of the gene with the cro ATG start codon, downstream of the strong PR promoter of phage lambda. The domain has been purified and characterized in in vitro complementation assays. It possessed no mannitol phosphorylation activity itself but was able to restore the phosphoenolpyruvate-dependent phosphorylation activity of two EIImtl phosphorylation site mutants, lacking His-554 or Cys-384. The complementary N-terminal domain was also expressed. Membranes possessing IICmtl were unable to phosphorylate mannitol at the expense of phosphoenolpyruvate. However, when the membranes were combined with the purified C-terminal domain, mannitol phosphorylation activity was restored. Mannitol transport and phosphorylation were also restored in vivo when the two plasmids encoding the N- and C-terminal domains were expressed in the same cell. These data demonstrate the existence of structurally and functionally distinct domains in EIImtl: a cytoplasmic domain with phosphorylating activity and a membrane-bound N-terminal domain which, in the presence of the cytoplasmic domain, is able to actively transport and phosphorylate mannitol. The ability to separate, overproduce, and purify structurally stable, enzymatically active domains opens the way for 3D structural studies as well as complete kinetic analysis of the activities of the individual domains and their interactions.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
1
pubmed:volume
30
pubmed:geneSymbol
mtlA
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
9478-85
pubmed:dateRevised
2008-11-21
pubmed:meshHeading
pubmed:year
1991
pubmed:articleTitle
Cytoplasmic phosphorylating domain of the mannitol-specific transport protein of the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli: overexpression, purification, and functional complementation with the mannitol binding domain.
pubmed:affiliation
BISON Research Institute, University of Groningen, The Netherlands.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't