Crystal Structure of the Catalytic Domain of Drosophila β1,4-Galactosyltransferase-7

Ramakrishnan, B.; Qasba, Pradman K.

doi:10.1074/jbc.m109.099564

Cited by 20 publications

(37 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As shown Fig. 3 (10), which is strictly conserved among ␤4GalT7 proteins, as shown by multiple sequence alignment (see Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…In the crystal structure of bovine ␤4GalT1 and Drosophila ␤4GalT7, these two polypeptides have been found to be located in the vicinity of the donor substrate UDP-Gal that is common to the ␤4GalT enzymes (19). Amino acids specific to ␤4GalT7 sequences are found in "motif 6" that contains a new Mn 2ϩ -binding motif, HXH (10). "Motif 8" exhibits 5 out of the 9 positions highly conserved in the ␤4GalT7 family, including two Cys residues forming a disulfide bond, as shown in Drosophila (Cys 300 -Cys 308 , corresponding to Cys 316 -Cys 324 in human sequence).…”

Section: Resultsmentioning

confidence: 99%

“…The mechanism of most inverting glycosyltransferases requires one acidic amino acid that activates the acceptor hydroxyl group by deprotonation (42). In Drosophila ␤4GalT7, Asp 211 (equivalent to Asp 228 in human ␤4GalT7) has been suggested to act as the catalytic base (10). In bovine ␤4GalT1, Asp 318 and possibly Glu 317 (equivalent to Asp 228 and Glu 227 in human ␤4GalT7) have been proposed as possible candidates to play such a role (40,41).…”

Section: Tablementioning

confidence: 99%

“…Interestingly, these motifs are also conserved between members of the ␤1,4-glycosyltransferase family, including seven ␤1,4-galactosyltransferases (␤1,4GalTs), two ␤1,4GalNAcTs, and four chondroitin synthases (19). Human ␤4GalT7 presents 73% protein sequence similarity with its Drosophila ortholog (10), and the seven human ␤4GalT proteins exhibit 25-55% sequence homology (19).…”

mentioning

confidence: 99%

“…In the invertebrate Drosophila melanogaster, a reverse genetic approach proved ␤4GalT7 to be essential for viability (9). The structure of the fly protein has recently been solved by x-ray crystallography (10). In Caenorhabditis elegans, defective ␤4GalT7 provokes serious morphological abnormalities during embryonic development (11).…”

mentioning

confidence: 99%

See 4 more Smart Citations

Identification of Key Functional Residues in the Active Site of Human β1,4-Galactosyltransferase 7

Talhaoui

Bui

Oriol

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

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.

show abstract

“…As shown Fig. 3 (10), which is strictly conserved among ␤4GalT7 proteins, as shown by multiple sequence alignment (see Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Tablementioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Identification of Key Functional Residues in the Active Site of Human β1,4-Galactosyltransferase 7

Talhaoui

Bui

Oriol

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

In Vitro Folding of β-1,4Galactosyltransferase and Polypeptide-α-N-Acetylgalactosaminyltransferase from the Inclusion Bodies

Ramakrishnan

Qasba

2013

Methods in Molecular Biology

View full text Add to dashboard Cite

The aim of this article is to present a unique in vitro folding technique for glycosyltransferases to generate active proteins that can be used for X-ray crystallographic and bioconjugation protocols. Although a number of in vitro refolding methods are available, β1,4galactosyltransferases in large quantities can be made using the current protocol only. This technique is not only limited to glycosyltransferases alone but has been successfully used to refold single-chain antibodies and other molecules. Although this in vitro folding method is quite similar to other methods, it differs from them by the use of S-sulfonation of the inclusion bodies before setting up the in vitro refolding of the protein.

show abstract

Investigations on β1,4-galactosyltransferase I using 6-sulfo-GlcNAc as an acceptor sugar substrate

et al. 2013

View full text Add to dashboard Cite

6-sulfate modified N-acetylglucosamine (6-sulfo-GlcNAc) is often found as part of many biologically important carbohydrate epitopes such as 6-sulfo-Le(X). In these epitopes, the 6-sulfo-GlcNAc moiety is extended by a galactose sugar in a β1-4 linkage. The β4GalT1 enzyme transfers galactose (Gal) from UDP-Gal to N-acetylglucosamine (GlcNAc) in the presence of manganese. Here we report that the β4GalT1 enzyme transfers Gal to the 6-sulfo-GlcNAc and 4-methylumbelliferyl-6-sulfo-N-acetyl-β-D-glucosaminide (6-sulfo-βGlcNAc-MU) acceptor substrates, although with very low efficiency. To understand the effect that the 6-sulfate group on the GlcNAc acceptor has on the catalytic activity of the β4GalT1 molecule, we have determined the crystal structure of the catalytic domain of bovine β4GalT1 mutant enzyme M344H-β4GalT1 complex with the 6-sulfo-GlcNAc molecule. In the crystal structure, the 6-sulfo-GlcNAc is bound to the protein in a way that is similar to the GlcNAc molecule. However, the 6-sulfate group engages in additional interactions with the hydrophobic region, residues 276-285, of the protein molecule, and this group is found wedged between the aromatic side chains of Phe-280 and Trp314 residues. Since the side chain of the Trp314 residue undergoes conformational changes during the catalytic cycle of the enzyme, molecular interaction between Trp314 and the 6-sulfate group might hinder this conformational change. Therefore, the lack of a favorable binding environment, together with hindrance to the conformational changes, might be responsible for the poor catalytic activity.

show abstract

Crystal Structure of the Catalytic Domain of Drosophila β1,4-Galactosyltransferase-7

Cited by 20 publications

References 43 publications

Identification of Key Functional Residues in the Active Site of Human β1,4-Galactosyltransferase 7

Identification of Key Functional Residues in the Active Site of Human β1,4-Galactosyltransferase 7

In Vitro Folding of β-1,4Galactosyltransferase and Polypeptide-α-N-Acetylgalactosaminyltransferase from the Inclusion Bodies

Investigations on β1,4-galactosyltransferase I using 6-sulfo-GlcNAc as an acceptor sugar substrate

Contact Info

Product

Resources

About