Effect of the Met344His mutation on the conformational dynamics of bovine beta-1,4-galactosyltransferase: Crystal structure of the Met344His mutant in complex with chitobiose

beta-1,4-Galactosyltransferase (beta4Gal-T1) in the presence of manganese ion transfers galactose from UDP-galactose (UDP-Gal) to N-acetylglucosamine (GlcNAc) that is either free or linked to an oligosaccharide. Crystallographic studies on bovine beta4Gal-T1 have shown that the primary metal binding site is located in the hinge region of a long flexible loop, which upon Mn2+ and UDP-Gal binding changes from an open to a closed conformation. This conformational change creates an oligosaccharide binding site in the enzyme. Neither UDP nor UDP analogues efficiently induce these conformational changes in the wild-type enzyme, thereby restricting the structural analysis of the acceptor binding site. The binding of Mn2+ involves an uncommon coordination to the Sdelta atom of Met344; when it is mutated to His, the mutant M344H, in the presence of Mn2+ and UDP-hexanolamine, readily changes to a closed conformation, facilitating the structural analysis of the enzyme bound with an oligosaccharide acceptor. Although the mutant M344H loses 98% of its Mn2+-dependent activity, it exhibits 25% of its activity in the presence of Mg2+. The crystal structures of M344H-Gal-T1 in complex with either UDP-Gal.Mn2+ or UDP-Gal.Mg2+, determined at 2.3 Angstrom resolution, show that the mutant enzyme in these complexes is in a closed conformation, and the coordination stereochemistry of Mg2+ is quite similar to that of Mn2+. Although either Mn2+ or Mg2+, together with UDP-Gal, binds and changes the conformation of the M344H mutant to the closed one, it is the Mg2+ complex that engages efficiently in catalyses. Thus, this property enabled us to crystallize the M344H mutant for the first time with the acceptor substrate chitobiose in the presence of UDP-hexanolamine and Mn2+. The crystal structure determined at 2.3 Angstrom resolution reveals that the GlcNAc residue at the nonreducing end of chitobiose makes extensive hydrophobic interactions with the highly conserved Tyr-286 residue

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