Linked Life Data

11479276

Source:http://linkedlifedata.com/resource/pubmed/id/11479276

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
8
pubmed:dateCreated
2001-7-31
pubmed:abstractText
The 4-amino-6-deoxy-monosaccharide D-perosamine is an important element in the glycosylation of interesting cell products, such as antibiotics and lipopolysaccharides (LPS) of Gram-positive and Gram-negative bacteria. The biosynthetic pathway of the precursor molecule, GDP-D-perosamine, in Vibrio cholerae O1 starts with an isomerisation of fructose-6-phosphate catalyzed by the bifunctional enzyme phosphomannose isomerase-guanosine diphosphomannose pyrophosphorylase (RfbA; E.C. 2.7.7.22) creating the intermediate mannose-6-phosphate, which is subsequently converted by the phosphomanno-mutase (RfbB; E.C. 5.4.2.8) and further by RfbA to GDP-D-mannose, to GDP-4 keto-6-deoxymannose by a 4,6-dehydratase (RfbD; E.C. 4.2.1.47) and finally to GDP-D-perosamine by an aminotransferase (RfbE; E.C. not yet classified). We cloned the rfbD and the rfbE genes of V. cholerae O1 in Escherichia coli expression vectors. Both biosynthetic enzymes were overproduced in E. coli BL21 (DE3) and their activities were analyzed. The enzymatic conversion from GDP-D-mannose to GDP-D-perosamine was optimized and the final product, GDP-D-perosamine, was purified and identified by nuclear magnetic resonance, mass spectrometry, and chromatography. The catalytically active form of the GDP-4-keto-6-deoxy-D-mannose-4-aminotransferase seems to be a tetramer of 170 kDa. The His-tag RfbE fusion protein has a Km of 0.06 mM and a Vmax value of 38 nkat/mg protein for the substrate GDP-4-keto-6-deoxy-D-mannose. The Km and Vmax values for the cosubstrate L-glutamate were 0.1 mM and 42 nkat/mg protein, respectively. The intention of this work is to establish a basis for both the in vitro production of GDP-D-perosamine and for an in vivo perosaminylation system in a suitable bacterial host, preferably E. coli.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
0959-6658
pubmed:author
pubmed:issnType
Print
pubmed:volume
11
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
655-61
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2001
pubmed:articleTitle
Expression and identification of the RfbE protein from Vibrio cholerae O1 and its use for the enzymatic synthesis of GDP-D-perosamine.
pubmed:affiliation
Chemische Mikrobiologie, Bergische Universität GH Wuppertal, Gauss-Str. 20, D-42097, Germany.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't