PDBsum entry 5cql

Transferase

PDB id: 5cql

Contents

Protein chain

261 a.a.

Metals

_HG ×5

Waters ×126

PDB id:

5cql

Name:

Transferase

Title:

Gtb mutant with mercury - e303a

Structure:

Histo-blood group abo system transferase. Chain: x. Fragment: catalytic domain (unp residues 64-354). Synonym: fucosylglycoprotein 3-alpha-galactosyltransferase, fucosylglycoprotein alpha-n-acetylgalactosaminyltransferase, glycoprotein-fucosylgalactoside alpha-n- acetylgalactosaminyltransferase,glycoprotein-fucosylgalactoside alpha-galactosyltransferase,histo-blood group a transferase,a transferase,histo-blood group b transferase,b transferase,nagat.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: abo. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.69Å R-factor: 0.176 R-free: 0.207

Authors:

S.M.L.Gagnon,R.J.Blackler

Key ref:

R.J.Blackler et al. (2017). Glycosyltransfer in mutants of putative catalytic residue Glu303 of the human ABO(H) A and B blood group glycosyltransferases GTA and GTB proceeds through a labile active site. Glycobiology, 27, 370-380. PubMed id: 27979997 DOI: 10.1093/glycob/cww117

Date:

22-Jul-15 Release date: 26-Oct-16

Protein chain

P16442  (BGAT_HUMAN) -  Histo-blood group ABO system transferase from Homo sapiens

Seq: 354 a.a.

Struc: 261 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

Enzyme reactions

• Enzyme class 1: E.C.2.4.1.37 - fucosylgalactoside 3-alpha-galactosyltransferase.
• Reaction: an alpha-L-fucosyl-(1->2)-beta-D-galactosyl derivative + UDP-alpha-D- galactose = an alpha-D-galactosyl-(1->3)-[alpha-L-fucosyl-(1->2)]-beta-D- galactosyl derivative + UDP + H⁺
• Enzyme class 2: E.C.2.4.1.40 - glycoprotein-fucosylgalactoside alpha-N-acetylgalactosaminyltransferase.
• Reaction: an alpha-L-fucosyl-(1->2)-beta-D-galactosyl derivative + UDP-N-acetyl- alpha-D-galactosamine = an N-acetyl-alpha-D-galactosaminyl-(1->3)-[alpha- L-fucosyl-(1->2)]-beta-D-galactosyl derivative + 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

Added reference

DOI no: 10.1093/glycob/cww117

Glycobiology 27:370-380 (2017)

PubMed id: 27979997

Glycosyltransfer in mutants of putative catalytic residue Glu303 of the human ABO(H) A and B blood group glycosyltransferases GTA and GTB proceeds through a labile active site.

R.J.Blackler, S.M.Gagnon, R.Polakowski, N.L.Rose, R.B.Zheng, J.A.Letts, A.R.Johal, B.Schuman, S.N.Borisova, M.M.Palcic, S.V.Evans.

ABSTRACT

The homologous glycosyltransferases α-1,3-N-acetylgalactosaminyltransferase (GTA) and α-1,3-galactosyltransferase (GTB) carry out the final synthetic step of the closely related human ABO(H) blood group A and B antigens. The catalytic mechanism of these model retaining enzymes remains under debate, where Glu303 has been suggested to act as a putative nucleophile in a double displacement mechanism, a local dipole stabilizing the intermediate in an orthogonal associative mechanism or a general base to stabilize the reactive oxocarbenium ion-like intermediate in an SNi-like mechanism. Kinetic analysis of GTA and GTB point mutants E303C, E303D, E303Q and E303A shows that despite the enzymes having nearly identical sequences, the corresponding mutants of GTA/GTB have up to a 13-fold difference in their residual activities relative to wild type. High-resolution single crystal X-ray diffraction studies reveal, surprisingly, that the mutated Cys, Asp and Gln functional groups are no more than 0.8 Å further from the anomeric carbon of donor substrate compared to wild type. However, complicating the analysis is the observation that Glu303 itself plays a critical role in maintaining the stability of a strained "double-turn" in the active site through several hydrogen bonds, and any mutation other than E303Q leads to significantly higher thermal motion or even disorder in the substrate recognition pockets. Thus, there is a remarkable juxtaposition of the mutants E303C and E303D, which retain significant activity despite disrupted active site architecture, with GTB/E303Q, which maintains active site architecture but exhibits zero activity. These findings indicate that nucleophilicity at position 303 is more catalytically valuable than active site stability and highlight the mechanistic elasticity of these enzymes.