1TW1

beta-1,4-galactosyltransferase mutant Met344His (m344H-Gal-T1) complex with UDP-galactose and magnesium

Summary for 1TW1

• Entry DOI: 10.2210/pdb1tw1/pdb
• Related: 1O0R 1TVY 1TW5
• Descriptor: Beta-1,4-galactosyltransferase 1, GALACTOSE-URIDINE-5'-DIPHOSPHATE, MAGNESIUM ION, ... (7 entities in total)
• Functional Keywords: met344his mutation; closed conformation; mn binding, transferase
• Biological source: Bos taurus (cattle)
• Cellular location: Isoform Long: Golgi apparatus, Golgi stack membrane; Single-pass type II membrane protein. Isoform Short: Golgi apparatus, Golgi stack membrane; Single-pass type II membrane protein. Processed beta-1,4-galactosyltransferase 1: Secreted: P08037
• Total number of polymer chains: 2
• Total formula weight: 67746.04

Authors

Ramakrishnan, B.,Boeggeman, E.,Qasba, P.K. (deposition date: 2004-06-30, release date: 2004-12-14, Last modification date: 2024-11-06)

Primary citation

Ramakrishnan, B.,Boeggeman, E.,Qasba, P.K.
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
Biochemistry, 43:12513-12522, 2004

PubMed Abstract: 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 Mn(2+) 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 Mn(2+) involves an uncommon coordination to the Sdelta atom of Met344; when it is mutated to His, the mutant M344H, in the presence of Mn(2+) 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 Mn(2+)-dependent activity, it exhibits 25% of its activity in the presence of Mg(2+). The crystal structures of M344H-Gal-T1 in complex with either UDP-Gal.Mn(2+) or UDP-Gal.Mg(2+), determined at 2.3 A resolution, show that the mutant enzyme in these complexes is in a closed conformation, and the coordination stereochemistry of Mg(2+) is quite similar to that of Mn(2+). Although either Mn(2+) or Mg(2+), together with UDP-Gal, binds and changes the conformation of the M344H mutant to the closed one, it is the Mg(2+) 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 Mn(2+). The crystal structure determined at 2.3 A resolution reveals that the GlcNAc residue at the nonreducing end of chitobiose makes extensive hydrophobic interactions with the highly conserved Tyr286 residue.

PubMed: 15449940
DOI: 10.1021/bi049007+

Experimental method

X-RAY DIFFRACTION (2.3 Å)