PDBsum entry 2fyc

Transferase

PDB id: 2fyc

Contents

Protein chains

123 a.a. *

272 a.a. *

Ligands

MES

GDU ×2

UDP ×2

Metals

_CA ×4

Waters ×765

* Residue conservation analysis

PDB id:

2fyc

Name:

Transferase

Title:

Crystal structure of the catalytic domain of bovine beta1,4- galactosyltransferase-i in complex with alpha-lactalbumin, ca and udp-galactose

Structure:

Alpha-lactalbumin. Chain: a, c. Synonym: lactose synthase b protein. Engineered: yes. Mutation: yes. Beta-1,4-galactosyltransferase. Chain: b, d. Fragment: residues 57-329. Engineered: yes

Source:

Mus musculus. House mouse. Organism_taxid: 10090. Gene: lalba. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693. Bos taurus. Cattle. Organism_taxid: 9913.

Biol. unit:

Dimer (from PQS)

Resolution:

2.00Å R-factor: 0.200 R-free: 0.246

Authors:

B.Ramakrishnan,V.Ramasamy,P.K.Qasba

Key ref:

B.Ramakrishnan et al. (2006). Structural snapshots of beta-1,4-galactosyltransferase-I along the kinetic pathway. J Mol Biol, 357, 1619-1633. PubMed id: 16497331 DOI: 10.1016/j.jmb.2006.01.088

Date:

07-Feb-06 Release date: 14-Mar-06

Protein chains

P29752  (LALBA_MOUSE) -  Alpha-lactalbumin from Mus musculus

Seq: 143 a.a.

Struc: 123 a.a.

Protein chains

P08037  (B4GT1_BOVIN) -  Beta-1,4-galactosyltransferase 1 from Bos taurus

Seq: 402 a.a.

Struc: 272 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class 1: Chains B, D: E.C.2.4.1.- - ?????
• Enzyme class 2: Chains B, D: E.C.2.4.1.22 - lactose synthase.
• Reaction: D-glucose + UDP-alpha-D-galactose = lactose + UDP + H⁺

D-glucose + UDP-alpha-D-galactose = lactose + UDP + H(+) (Bound ligand (Het Group name = GDU) matches with 69.44% similarity)

• Enzyme class 3: Chains B, D: E.C.2.4.1.275 - neolactotriaosylceramide beta-1,4-galactosyltransferase.
• Reaction: a beta-D-GlcNAc-(1->3)-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1)- Cer(d18:1(4E)) + UDP-alpha-D-galactose = a neolactoside nLc4Cer(d18:1(4E)) + UDP + H⁺

beta-D-GlcNAc-(1->3)-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1)- Cer(d18:1(4E)) + UDP-alpha-D-galactose = neolactoside nLc4Cer(d18:1(4E)) + UDP + H(+) (Bound ligand (Het Group name = GDU) matches with 69.44% similarity)

• Enzyme class 4: Chains B, D: E.C.2.4.1.38 - beta-N-acetylglucosaminylglycopeptide beta-1,4-galactosyltransferase.
• Reaction: an N-acetyl-beta-D-glucosaminyl derivative + UDP-alpha-D-galactose = a beta-D-galactosyl-(1->4)-N-acetyl-beta-D-glucosaminyl derivative + UDP + H⁺

N-acetyl-beta-D-glucosaminyl derivative (Bound ligand (Het Group name = UDP) matches with 42.86% similarity) + UDP-alpha-D-galactose = beta-D-galactosyl-(1->4)-N-acetyl-beta-D-glucosaminyl derivative + UDP + H(+) (Bound ligand (Het Group name = GDU) matches with 69.44% similarity)

• Enzyme class 5: Chains B, D: E.C.2.4.1.90 - N-acetyllactosamine synthase.
• Reaction: N-acetyl-D-glucosamine + UDP-alpha-D-galactose = beta-D-galactosyl- (1->4)-N-acetyl-D-glucosamine + UDP + H⁺

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1016/j.jmb.2006.01.088

J Mol Biol 357:1619-1633 (2006)

PubMed id: 16497331

Structural snapshots of beta-1,4-galactosyltransferase-I along the kinetic pathway.

B.Ramakrishnan, V.Ramasamy, P.K.Qasba.

ABSTRACT

During the catalytic cycle of beta1,4-galactosyltransferase-1 (Gal-T1), upon the binding of Mn(2+) followed by UDP-Gal, two flexible loops, a long and a short loop, change their conformation from open to closed. We have determined the crystal structures of a human M340H-Gal-T1 mutant in the open conformation (apo-enzyme), its Mn(2+) and Mn(2+)-UDP-Gal-bound complexes, and of a pentenary complex of bovine Gal-T1-Mn(2+)-UDP-GalNAc-Glc-alpha-lactalbumin. These studies show that during the conformational changes in Gal-T1, the coordination of Mn(2+) undergoes significant changes. It loses a coordination bond with a water molecule bound in the open conformation of Gal-T1 while forming a new coordination bond with another water molecule in the closed conformation, creating an active ground-state structure that facilitates enzyme catalysis. In the crystal structure of the pentenary complex, the N-acetylglucosamine (GlcNAc) moiety is found cleaved from UDP-GalNAc and is placed 2.7A away from the O4 oxygen atom of the acceptor Glc molecule, yet to form the product. The anomeric C1 atom of the cleaved GalNAc moiety has only two covalent bonds with its non-hydrogen atoms (O5 and C2 atoms), similar to either an oxocarbenium ion or N-acetylgalactal form, which are crystallographically indistinguishable at the present resolution. The structure also shows that the newly formed, metal-coordinating water molecule forms a hydrogen bond with the beta-phosphate group of the cleaved UDP moiety. This hydrogen bond formation results in the rotation of the beta-phosphate group of UDP away from the cleaved GalNAc moiety, thereby preventing the re-formation of the UDP-sugar during catalysis. Therefore, this water molecule plays an important role during catalysis in ensuring that the catalytic reaction proceeds in a forward direction.

Selected figure(s)

Figure 1.
Figure 1. The schematic diagram showing the kinetic pathway of the Gal-T1 (GT) enzyme and of lactose synthase reaction where, in the presence of aLA, glucose (Glc) is the acceptor substrate. The crystal structures of the representative intermediates determined here along the reaction pathway, together with a previously determined structure, are indicated underneath the reaction scheme with the corresponding Figures here, in blue and red, respectively. First the apo-enzyme exists in an open conformation (Figure 2(a)), to which the manganese ion (Mn2+) binds (Figure 3(a)), followed by the donor substrate, UDP-Gal (Figure 3(b)). Upon UDP-Gal or UDP-sugar binding the enzyme undergoes conformational changes from open to closed (Figure 3(c)), creating the acceptor and aLA binding sites. aLA and Glc bind together synergistically to GT-Mn2+-UDP-sugar complex in the closed conformation, forming a ground state pentenary complex (Figure 4). During the transition state the sugar moiety is cleaved from UDP-sugar and exists as an oxocarbenium ion, shown as Gal* (or GalNAc* in Figure 4), which forms a disaccharide linkage with the acceptor sugar, Glc, and is then released from the enzyme (GT) along with the aLA molecule from the pentenary complex. Here, we have used UDP-GalNAc as the donor substrate to crystallize the pentenary complex (Figure 4), since due to the steric hindrance caused by the side-chain of Tyr286 residue with the N-acetyl moiety of UDP-GalNAc, the transfer of GalNAc from UDP-GalNAc to Glc is very poor, thus enabling us to crystallize the pentenary complex.

Figure 6.
Figure 6. Metal ion-bound water molecule observed in the crystal structures of nucleotide or sugar nucleotide-bound complexes of other glycosyltransferases. It seems that in Gal-T1, the presence of the metal ion-bound water molecule, W5, is important for the rotation of the b-phosphate oxygen atoms to form a hydrogen bond with the O1 oxygen atom, which ensures that the catalytic reaction proceeds. Since cleavage of the sugar moiety from the nucleotide sugar is a common step in all the glycosyltransferases, irrespective of their catalytic mechanism, the presence of a metal-bound water molecule in the vicinity of the glycosidic bond of the bound nucleotide-sugar may be a common structural feature. We have examined the (a) b1,2-N-acetylglucosaminyltransferase (1FOA.PDB), (b) b1,3-glucuronyltransferase I (1KWS.PDB), and (c) a1,4-N-acetylhexosaminyltransferase (1ON6.PDB), with their nucleotide-sugar complexes. In all these structures, a metal-bound water molecule is found in the vicinity of the glycosidic bond. Thus, this water molecule seems to play an important role in the catalytic mechanism, similar to the one (W5) found in the present crystal structures.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2006, 357, 1619-1633) copyright 2006.

Figures were selected by an automated process.