title: Structure-based design of beta 1,4-galactosyltransferase I (beta 4Gal-T1) with equally efficient N-acetylgalactosaminyltransferase activity: point mutation broadens beta 4Gal-T1 donor specificity.
abstract: beta1,4-Galactosyltransferase I (Gal-T1) normally transfers Gal from UDP-Gal to GlcNAc in the presence of Mn(2+) ion. In the presence of alpha-lactalbumin (LA), the Gal acceptor specificity is altered from GlcNAc to Glc. Gal-T1 also transfers GalNAc from UDP-GalNAc to GlcNAc, but with only approximately 0.1% of Gal-T activity. To understand this low GalNAc-transferase activity, we have carried out the crystal structure analysis of the Gal-T1.LA complex with UDP-GalNAc at 2.1-A resolution. The crystal structure reveals that the UDP-GalNAc binding to Gal-T1 is similar to the binding of UDP-Gal to Gal-T1, except for an additional hydrogen bond formed between the N-acetyl group of GalNAc moiety with the Tyr-289 side chain hydroxyl group. Elimination of this additional hydrogen bond by mutating Tyr-289 residue to Leu, Ile, or Asn enhances the GalNAc-transferase activity. Although all three mutants exhibit enhanced GalNAc-transferase activity, the mutant Y289L exhibits GalNAc-transferase activity that is nearly 100% of its Gal-T activity, even while completely retaining its Gal-T activity. The steady state kinetic analyses on the Leu-289 mutant indicate that the K(m) for GlcNAc has increased compared to the wild type. On the other hand, the catalytic constant (k(cat)) in the Gal-T reaction is comparable with the wild type, whereas it is 3-5-fold higher in the GalNAc-T reaction. Interestingly, in the presence of LA, these mutants also transfer GalNAc to Glc instead of to GlcNAc. The present study demonstrates that, in the Gal-T family, the Tyr-289/Phe-289 residue largely determines the sugar donor specificity.
authors.affiliation: Structural Glycobiology Section and Intramural Research Support Program-SAIC, Laboratory of Experimental and Computational Biology, Center for Cancer Research, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702, USA.
authors.forename: Boopathy
authors.initials: B
authors.lastname: Ramakrishnan
authors.forename: Pradman K
authors.initials: PK
authors.lastname: Qasba
mesh_terms: D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002417:Cattle; D017931:DNA Primers; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism*; D017354:Point Mutation; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C119701:beta-1,4-galactosyltransferase I