Linked Life Data

8663048

Source:http://linkedlifedata.com/resource/pubmed/id/8663048

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
26
pubmed:dateCreated
1996-8-20
pubmed:databankReference
pubmed:abstractText
An N-acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-NeuAc synthetase) was isolated from a Haemophilus ducreyi strain 35000 cell lysate and partially characterized. The enzyme catalyzes the reaction of CTP and NeuAc to form CMP-NeuAc, which is the nucleotide sugar donor used by sialyltransferases. Previous studies have shown that the outer membrane lipooligosaccharides of H. ducreyi contain terminal sialic acid attached to N-acetyllactosamine and that this modification is likely important to its pathogenesis. Therefore, to investigate the role of sialic acid in H. ducreyi pathogenesis, the gene encoding the CMP-NeuAc synthetase was cloned using degenerate oligonucleotide probes derived from NH2-terminal sequence data, and the nucleotide sequence was determined. The derived amino acid sequence of the CMP-NeuAc synthetase gene has homology to other CMP-NeuAc synthetases and to a lesser extent to CMP-2-keto-3-deoxy-D-manno-octulosonic acid synthetases. The gene was cloned into a T7 expression vector, the protein expressed in Escherichia coli, and purified to apparent homogeneity by anion exchange, Green 19 dye, and hydrophobic interaction chromatography. The final step yielded 20 mg of pure protein/liter of culture. The protein has a predicted molecular mass of 25440.6 Da, which was confirmed by electrospray mass spectrometry (Mexpt = 25439.9 +/- 1.4 Da). The enzyme appears to exist as a dimer by size exclusion chromatography. In contrast to other bacterial CMP-NeuAc synthetases, the H. ducreyi enzyme exhibited a different substrate specificity, being capable of also using N-glycolylneuraminic acid as a substrate.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
28
pubmed:volume
271
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
15373-80
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
Purification, cloning, and expression of a cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from Haemophilus ducreyi.
pubmed:affiliation
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't