Linked Life Data

20843813

Source:http://linkedlifedata.com/resource/pubmed/id/20843813

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
48
pubmed:dateCreated
2010-11-24
pubmed:abstractText
Glycosaminoglycans (GAGs) play a central role in many pathophysiological events, and exogenous xyloside substrates of ?1,4-galactosyltransferase 7 (?4GalT7), a major enzyme of GAG biosynthesis, have interesting biomedical applications. To predict functional peptide regions important for substrate binding and activity of human ?4GalT7, we conducted a phylogenetic analysis of the ?1,4-galactosyltransferase family and generated a molecular model using the x-ray structure of Drosophila ?4GalT7-UDP as template. Two evolutionary conserved motifs, (163)DVD(165) and (221)FWGWGREDDE(230), are central in the organization of the enzyme active site. This model was challenged by systematic engineering of point mutations, combined with in vitro and ex vivo functional assays. Investigation of the kinetic properties of purified recombinant wild-type ?4GalT7 and selected mutants identified Trp(224) as a key residue governing both donor and acceptor substrate binding. Our results also suggested the involvement of the canonical carboxylate residue Asp(228) acting as general base in the reaction catalyzed by human ?4GalT7. Importantly, ex vivo functional tests demonstrated that regulation of GAG synthesis is highly responsive to modification of these key active site amino acids. Interestingly, engineering mutants at position 224 allowed us to modify the affinity and to modulate the specificity of human ?4GalT7 toward UDP-sugars and xyloside acceptors. Furthermore, the W224H mutant was able to sustain decorin GAG chain substitution but not GAG synthesis from exogenously added xyloside. Altogether, this study provides novel insight into human ?4GalT7 active site functional domains, allowing manipulation of this enzyme critical for the regulation of GAG synthesis. A better understanding of the mechanism underlying GAG assembly paves the way toward GAG-based therapeutics.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
1083-351X
pubmed:author
pubmed:issnType
Electronic
pubmed:day
26
pubmed:volume
285
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
37342-58
pubmed:dateRevised
2011-1-6
pubmed:meshHeading
pubmed:year
2010
pubmed:articleTitle
Identification of key functional residues in the active site of human {beta}1,4-galactosyltransferase 7: a major enzyme in the glycosaminoglycan synthesis pathway.
pubmed:affiliation
Faculté de Médecine, UMR 7561 CNRS-Université de Nancy I, BP 184, 54505 Vandoeuvre-lès-Nancy, France.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't