Linked Life Data

16303119

Source:http://linkedlifedata.com/resource/pubmed/id/16303119

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2005-12-19
pubmed:abstractText
The putative beta-glucuronidase from Thermotoga maritima, comprising 563 amino acid residues conjugated with a Hisx6 tag, was cloned and expressed in Escherichia coli. The enzyme has a moderately broad specificity, hydrolysing a range of p-nitrophenyl glycoside substrates, but has greatest activity on p-nitrophenyl beta-D-glucosiduronic acid (kcat=68 s(-1), kcat/K(M)= 4.5x10(5) M(-1) s(-1)). The enzyme also shows a relatively broad pH-dependence with activity from pH4.5 to 7.5 and a maximum at pH6.5. As expected the enzyme is stable towards heat denaturation, with a half life of 3h at 85 degrees C, in contrast to the mesophilic E. coli enzyme, which has a half life of 2.6h at 50 degrees C. The identity of the catalytic nucleophile was confirmed as Glu476 within the sequence VTEFGAD by trapping the glycosyl-enzyme intermediate using the mechanism-based inactivator, 2-deoxy-2-fluoro-beta-D-glucosyluronic acid fluoride and identifying the labeled peptide in peptic digests by HPLC-MS/MS methodologies. Consistent with this, the Glu476Ala mutant was shown to be hydrolytically inactive. The acid/base catalyst was confirmed as Glu383 by generation and kinetic analysis of enzyme mutants modified at that position, Glu383Ala and Glu383Gln. The demonstration of activity rescue by azide is consistent with the proposed role for this residue. This enzyme therefore appears suitable for use in enzymatic oligosaccharide synthesis in either the transglycosylation mode or by use of glycosynthase and thioglycoligase approaches.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0008-6215
pubmed:author
pubmed:issnType
Print
pubmed:day
16
pubmed:volume
341
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
49-59
pubmed:dateRevised
2008-11-21
pubmed:meshHeading
pubmed:year
2006
pubmed:articleTitle
Cloning and characterization of Thermotoga maritima beta-glucuronidase.
pubmed:affiliation
Protein Engineering Network of Centres of Excellence of Canada, Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't