Identification of key functional residues in the active site of human {beta}1,4-galactosyltransferase 7: a major enzyme in the glycosaminoglycan synthesis pathway.

ABSTRACT: 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.

SUBMITTER: Talhaoui I 

PROVIDER: S-EPMC2988340 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature