PDBsum entry 1vzt

Transferase

PDB id: 1vzt

Contents

Protein chains

287 a.a. *

Ligands

UDP ×2

TRS ×2

Metals

_MN ×2

Waters ×442

* Residue conservation analysis

PDB id:

1vzt

Name:

Transferase

Title:

Roles of individual residues of alpha-1,3 galactosyltransferases in substrate binding and catalysis

Structure:

N-acetyllactosaminide alpha-1,3-galactosyltransferase. Chain: a, b. Fragment: catalytic domain, residues 80-368. Synonym: galactosyltransferase, udp-galactose, beta-d-galactosyl-1,4- n-acetyl-d-glucosaminide alpha-1,3-galactosyltransferase. Engineered: yes. Mutation: yes

Source:

Bos taurus. Bovine. Organism_taxid: 9913. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_variant: dh5-alpha.

Resolution:

2.00Å R-factor: 0.193 R-free: 0.220

Authors:

Y.Zhang,G.J.Swaminathan,A.Deshpande,R.Natesh,X.Xie,K.R.Acharya,K.Brew

Key ref:

Y.Zhang et al. (2003). Roles of individual enzyme-substrate interactions by alpha-1,3-galactosyltransferase in catalysis and specificity. Biochemistry, 42, 13512-13521. PubMed id: 14621997 DOI: 10.1021/bi035430r

Date:

26-May-04 Release date: 26-May-05

Supersedes:

1o7r

Protein chains

P14769  (GGTA1_BOVIN) -  N-acetyllactosaminide alpha-1,3-galactosyltransferase from Bos taurus

Seq: 368 a.a.

Struc: 287 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.2.4.1.87 - N-acetyllactosaminide 3-alpha-galactosyltransferase.
• Reaction: a beta-D-galactosyl-(1->4)-N-acetyl-beta-D-glucosaminyl derivative + UDP- alpha-D-galactose = an alpha-D-galactosyl-(1->3)-beta-D-galactosyl- (1->4)-N-acetyl-beta-D-glucosaminyl derivative + UDP + H⁺

beta-D-galactosyl-(1->4)-N-acetyl-beta-D-glucosaminyl derivative + UDP- alpha-D-galactose = alpha-D-galactosyl-(1->3)-beta-D-galactosyl- (1->4)-N-acetyl-beta-D-glucosaminyl derivative + UDP + H(+) (Bound ligand (Het Group name = UDP) corresponds exactly)

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

Key reference

DOI no: 10.1021/bi035430r

Biochemistry 42:13512-13521 (2003)

PubMed id: 14621997

Roles of individual enzyme-substrate interactions by alpha-1,3-galactosyltransferase in catalysis and specificity.

Y.Zhang, G.J.Swaminathan, A.Deshpande, E.Boix, R.Natesh, Z.Xie, K.R.Acharya, K.Brew.

ABSTRACT

The retaining glycosyltransferase, alpha-1,3-galactosyltransferase (alpha3GT), is mutationally inactivated in humans, leading to the presence of circulating antibodies against its product, the alpha-Gal epitope. alpha3GT catalyzes galactose transfer from UDP-Gal to beta-linked galactosides, such as lactose, and in the absence of an acceptor substrate, to water at a lower rate. We have used site-directed mutagenesis to investigate the roles in catalysis and specificity of residues in alpha3GT that form H-bonds as well as other interactions with substrates. Mutation of the conserved Glu(317) to Gln weakens lactose binding and reduces the k(cat) for galactosyltransfer to lactose and water by 2400 and 120, respectively. The structure is not perturbed by this substitution, but the orientation of the bound lactose molecule is changed. The magnitude of these changes does not support a previous proposal that Glu(317) is the catalytic nucleophile in a double displacement mechanism and suggests it acts in acceptor substrate binding and in stabilizing a cationic transition state for cleavage of the bond between UDP and C1 of the galactose. Cleavage of this bond also linked to a conformational change in the C-terminal region of alpha3GT that is coupled with UDP binding. Mutagenesis indicates that His(280), which is projected to interact with the 2-OH of the galactose moiety of UDP-Gal, is a key residue in the stringent donor substrate specificity through its role in stabilizing the bound UDP-Gal in a suitable conformation for catalysis. Mutation of Gln(247), which forms multiple interactions with acceptor substrates, to Glu reduces the catalytic rate of galactose transfer to lactose but not to water. This mutation is predicted to perturb the orientation or environment of the bound acceptor substrate. The results highlight the importance of H-bonds between enzyme and substrates in this glycosyltransferase, in arranging substrates in appropriate conformations and orientation for efficient catalysis. These factors are manifested in increases in catalytic rate rather than substrate affinity.