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Frederick, MD, United States

Ramakrishnan B.,Center for Cancer Research Nanobiology Program | Ramakrishnan B.,SAIC | Qasba P.K.,Center for Cancer Research Nanobiology Program
Journal of Biological Chemistry | Year: 2010

The β1,4-galactosyltransferase-7 (β4Gal-T7) enzyme, one of seven members of the β4Gal-T family, transfers in the presence of manganese Gal from UDP-Gal to an acceptor sugar (xylose) that is attached to a side chain hydroxyl group of Ser/Thr residues of proteoglycan proteins. It exhibits the least protein sequence similarity with the other family members, including the well studied family member β4Gal-T1, which, in the presence of manganese, transfers Gal from UDP-Gal to GlcNAc. We report here the crystal structure of the catalytic domain of β4Gal-T7 from Drosophila in the presence of manganese and UDP at 1.81 Å resolution. In the crystal structure, a new manganese ion-binding motif (HXH) has been observed. Superposition of the crystal structures of β4Gal-T7 and β4Gal-T1 shows that the catalytic pocket and the substrate-binding sites in these proteins are similar. Compared with GlcNAc, xylose has a hydroxyl group (instead of anN-acetyl group) at C2 and lacks theCH2OH group at C5; thus, these protein structures show significant differences in their acceptor-binding site. Modeling of xylose in the acceptor-binding site of the β4Gal-T7 crystal structure shows that the aromatic side chain of Tyr177 interacts strongly with the C5 atom of xylose, causing steric hindrance to any additional group at C5. Because Drosophila Cd7 has a 73% protein sequence similarity to human Cd7, the present crystal structure offers a structure-based explanation for the mutations in human Cd7 that have been linked to Ehlers-Danlos syndrome. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Pasek M.,Center for Cancer Research Nanobiology Program | Ramakrishnan B.,Center for Cancer Research Nanobiology Program | Ramakrishnan B.,SAIC | Boeggeman E.,Center for Cancer Research Nanobiology Program | And 5 more authors.
Glycobiology | Year: 2012

In recent years, sugars with a unique chemical handle have been used to detect and elucidate the function of glycoconjugates. Such chemical handles have generally been part of an N-acetyl moiety of a sugar. We have previously developed several applications using the single mutant Y289L-β1,4- galactosyltransferase I (Y289L-β4Gal-T1) and the wild-type polypeptide-α-GalNAc-T enzymes with UDP-C2-keto-Gal. Here, we describe for the first time that the GlcNAc-transferring enzymes-R228K-Y289L-β4Gal-T1 mutant enzyme, the wild-type human β1,3-N-acetylglucosaminyltransferase-2 and human Maniac Fringe-can also transfer the GlcNAc analog C2-keto-Glc molecule from UDP-C2-keto-Glc to their respective acceptor substrates. Although the R228K-Y289L-β4Gal-T1 mutant enzyme transfers the donor sugar substrate GlcNAc or its analog C2-keto-Glc only to its natural acceptor substrate, GlcNAc, it does not transfer to its analog C2-keto-Glc. Thus, these observations suggest that the GlcNAc-transferring glycosyltransferases can generally accommodate a chemical handle in the N-acetyl-binding cavity of the donor sugar substrate, but not in the N-acetyl-binding cavity of the acceptor sugar. © The Author 2011. Published by Oxford University Press. All rights reserved.

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