PDBsum entry: 1pzy

Transferase activator/transferase

PDB id: 1pzy

Contents

Protein chains

123 a.a. *

271 a.a. *

Ligands

NAG ×2

UDP ×2

Metals

_CA ×2

_MN ×2

Waters ×251

* Residue conservation analysis

PDB id:

1pzy

Name:

Transferase activator/transferase

Title:

W314a-beta1,4-galactosyltransferase-i complexed with alpha-l in the presence of n-acetylglucosamine, udp and manganese

Structure:

Alpha-lactalbumin. Chain: a, c. Fragment: regulatory subunit of lactose synthase. Synonym: lactalbumin, alpha. Engineered: yes. Other_details: chains a and b form first, b and d second la synthase complex. Beta-1,4-galactosyltransferase. Chain: b, d.

Source:

Mus musculus. House mouse. Organism_taxid: 10090. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693. Bos taurus. Cattle. Organism_taxid: 9913. Other_details: n-terminal carries a tag containing 13 amino

Biol. unit:

Dimer (from PQS)

Resolution:

2.30Å R-factor: 0.207 R-free: 0.253

Authors:

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

Key ref:

V.Ramasamy et al. (2003). The role of tryptophan 314 in the conformational changes of beta1,4-galactosyltransferase-I. J Mol Biol, 331, 1065-1076. PubMed id: 12927542 DOI: 10.1016/S0022-2836(03)00790-3

Date:

14-Jul-03 Release date: 02-Sep-03

Protein chains

P29752  (LALBA_MOUSE) -  Alpha-lactalbumin

Seq: 143 a.a.

Struc: 123 a.a.

Protein chains

P08037  (B4GT1_BOVIN) -  Beta-1,4-galactosyltransferase 1

Seq: 402 a.a.

Struc: 271 a.a.*

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

Enzyme reactions

• Enzyme class 1: Chains B, D: E.C.2.4.1.22 - Lactose synthase.
• Reaction: UDP-galactose + D-glucose = UDP + lactose

UDP-galactose + D-glucose (Bound ligand (Het Group name = NAG) matches with 68.75% similarity) = UDP (Bound ligand (Het Group name = UDP) corresponds exactly) + lactose

• Enzyme class 2: Chains B, D: E.C.2.4.1.38 - Beta-N-acetylglucosaminylglycopeptide beta-1,4-galactosyltransferase.
• Reaction: UDP-galactose + N-acetyl-beta-D-glucosaminylglycopeptide = UDP + beta-D- galactosyl-(1->4)-N-acetyl-beta-D-glucosaminylglycopeptide

UDP-galactose + N-acetyl-beta-D-glucosaminylglycopeptide (Bound ligand (Het Group name = NAG) matches with 93.75% similarity) = UDP (Bound ligand (Het Group name = UDP) corresponds exactly) + beta-D- galactosyl-(1->4)-N-acetyl-beta-D-glucosaminylglycopeptide

• Enzyme class 3: Chains B, D: E.C.2.4.1.90 - N-acetyllactosamine synthase.
• Reaction: UDP-galactose + N-acetyl-D-glucosamine = UDP + N-acetyllactosamine

UDP-galactose + N-acetyl-D-glucosamine (Bound ligand (Het Group name = NAG) corresponds exactly) = UDP (Bound ligand (Het Group name = UDP) corresponds exactly) + N-acetyllactosamine

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

 Gene Ontology (GO) functional annotation 

• Cellular component: extracellular region (1 term)
• Biological process: carbohydrate metabolic process (2 terms)
• Biochemical function: protein binding (4 terms)

reference

DOI no: 10.1016/S0022-2836(03)00790-3

J Mol Biol 331:1065-1076 (2003)

PubMed id: 12927542

The role of tryptophan 314 in the conformational changes of beta1,4-galactosyltransferase-I.

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

ABSTRACT

beta1,4-Galactosyltransferase-I (beta4Gal-T1) undergoes critical conformational changes upon substrate binding from an open conformation (conf-I) to the closed conformation (conf-II). This change involves two flexible loops: the small (residues 313-316) and the long loop (residues 345-365). Upon substrate binding, Trp314 in the small flexible loop moves towards the catalytic pocket and interacts with the donor and the acceptor substrates. For a better understanding of the role played by Trp314 in the conformational changes of beta4Gal-T1, we mutated it to Ala and carried out substrate-binding, proteolytic and crystallographic studies. The W314A mutation reduces the enzymatic activity, binding to substrates and to the modifier protein, alpha-lactalbumin (LA), by over 99%. The limited proteolysis with Glu-C or Lys-C proteases shows differences in the rate of cleavage of the long loop of the wild-type and mutant W314A, indicating conformational differences in the region between the two proteins. Without substrate, the mutant crystallizes in a conformation (conf-I') (1.9A resolution crystal structure), that is not identical with, but close to an open conformation (conf-I), whereas its complex with the substrates and alpha-lactalbumin, crystallizes in a conformation (2.3A resolution crystal structure) that is identical with the closed conformation (conf-II). This study shows the crucial role Trp314 plays in the conformational state of the long loop, in the binding of substrates and in the catalytic mechanism of the enzyme.

Selected figure(s)

Figure 5.
Figure 5. The difference Fourier electron density omit maps for GlcNAc, UDP and Mn 2⁺ , contoured at the 2s level observed in the closed conformation found in the complex of W314A-b4Gal-T1 with LA and substrates. Also shown is the electron density of Asp318 that lies underneath the GlcNAc molecule. The carboxylate group of Asp318 hydrogen bonds with the hydroxyl group at C4 of GlcNAc. (Figures generated using Refs 18 and 19).

Figure 7.
Figure 7. (A) A representation of the enzymatic mechanism of b4Gal-T1. (I) Deprotonation of the nucleophilic hydroxyl group at C4 of GlcNAc by Asp318. (II) An oxo- carbenium ion transition state intermediate, where the galactose residue is in a half-boat confor- mation, with C10 in the carbenium ion state. (III) Final product, Lac- NAc, with galactose b- linked to GlcNAc. (B) Superposition of UDP- Gal and Mn 2⁺ in the closed con- formation of wild-type b4Gal-T1 (purple) (pdb code 1O0R) with the ligands UDP, Mn 2⁺ and GlcNAc in the W314A-b4Gal-T1-LA complex (green). The superposition of these two structures shows that the distance between O4 of GlcNAc and C10 of galactose is 3.8 A š .

The above figures are reprinted by permission from Elsevier: J Mol Biol (2003, 331, 1065-1076) copyright 2003.

Figures were selected by the author.