Lipopolysaccharide 3-alpha-galactosyltransferase

 

Lipo-polysaccharide galactosyl transferase (LGTC) is a retaining glycosyl transferase which catalyses the transfer of alpha-D-galactose to a terminal lactose during the synthesis of lipo-polysaccharides with retention of stereochemistry at the anomeric carbon. The enzyme held at the surface of the bacterial plasma membrane through electrostatic interaction between basic residues and phospho-lipids and interactions between hydrophobic regions.

 

Reference Protein and Structure

Sequence
Q93EK7 UniProt IPR002495 (Sequence Homologues) (PDB Homologues)
Biological species
Neisseria meningitidis (Bacteria) Uniprot
PDB
1ga8 - CRYSTAL STRUCTURE OF GALACOSYLTRANSFERASE LGTC IN COMPLEX WITH DONOR AND ACCEPTOR SUGAR ANALOGS. (2.0 Å) PDBe PDBsum 1ga8
Catalytic CATH Domains
3.90.550.10 CATHdb (see all for 1ga8)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:2.4.1.44)

beta-D-galactosyl group
CHEBI:16289ChEBI
+
UDP-alpha-D-galactose(2-)
CHEBI:66914ChEBI
→
alpha-D-galactosyl-(1->4)-beta-D-galactosyl group
CHEBI:139098ChEBI
+
UDP(3-)
CHEBI:58223ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: UDP-galactose:lipopolysaccharide alpha,3-galactosyltransferase, UDP-galactose:polysaccharide galactosyltransferase, UDPgalactose:lipopolysaccharide 3-alpha-D-galactosyltransferase, Uridine diphosphate galactose:lipopolysaccharide alpha-3-galactosyltransferase, Uridine diphosphogalactose-lipopolysaccharide alpha,3-galactosyltransferase, Lipopolysaccharide galactosyltransferase, Lipopolysaccharide 1,3-galactosyltransferase, UDP-galactose:lipopolysaccharide 3-alpha-D-galactosyltransferase,

Enzyme Mechanism

Introduction

It follows an SNi-type mechanism, similar to that of the OtsA enzyme. Lacking a strong nucleophile like other retaining enzymes in the glycosyltransferase 6 family (Gln189 is a poor nucleophile), O4 from lactose (galactose) directly attacks C1 on galactose in UDP-galactose front side via a dissociative SNi transition state. Proton transfer likely occurs from the 4'OH to O3 on the beta phosphate (UDP). The surrounding active site residues and Mn2+ ion help promote the front-side mechanism in the stabilisation of the reactant transition states. So far it is unclear whether a shortly lived intermediate like OtsA forms in the reaction and identify exactly the residues to stabilise the intermediate. Regardless, for LGTC it is far more favourable to proceed via a SNi mechanism, intermediate or not as opposed to the double displacement mechanism previously proposed.

Catalytic Residues Roles

UniProt PDB* (1ga8)
Lys250 Lys250A Lys250 out of all the residues has the largest electrostatic stabilisation effect by hydrogen bonds with the alpha and beta phosphates of the UDP leaving group. hydrogen bond donor, electrostatic stabiliser, polar interaction
Asp103, Asp105, His244 Asp103A, Asp105A, His244A Co-ordinate to Mn2+ ion which in turn stabilises the negatively charged phosphate, making UDP a good leaving group. metal ligand
Gln189 Gln189A Shown to have poor nucleophilicity, instead having a role in stabilising the developing charges during the transition state at the anomeric carbon in the reaction. electrostatic stabiliser, polar interaction
His78, Asp130, Asp188 His78A, Asp130A, Asp188A Form hydrogen bonds with the reactants to stabilise the transition state. hydrogen bond donor, electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

bimolecular nucleophilic substitution, proton transfer, overall product formed, overall reactant used

References

  1. Gómez H et al. (2012), J Am Chem Soc, 134, 4743-4752. Retaining glycosyltransferase mechanism studied by QM/MM methods: lipopolysaccharyl-α-1,4-galactosyltransferase C transfers α-galactose via an oxocarbenium ion-like transition state. DOI:10.1021/ja210490f. PMID:22352786.
  2. Persson K et al. (2001), Nat Struct Biol, 8, 166-175. Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs. DOI:10.1038/84168. PMID:11175908.

Step 1. Nucleophilic attack by 4'OH on glucose on the 1' anomeric C is facilitated by the protonation of the leaving group UDP.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp105A metal ligand
His244A metal ligand
Asp103A metal ligand
Gln189A electrostatic stabiliser
His78A hydrogen bond donor
Asp130A hydrogen bond acceptor
Asp188A hydrogen bond acceptor
His78A electrostatic stabiliser
Asp130A electrostatic stabiliser
Asp188A electrostatic stabiliser
Lys250A electrostatic stabiliser, hydrogen bond donor, polar interaction
Gln189A polar interaction

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, overall product formed, overall reactant used
Download: Image, Marvin File

Step 1. Nucleophilic attack by 4'OH on glucose on the 1' anomeric C is facilitated by the protonation of the leaving group UDP.

Products.

Introduction

Structural data and comparisons to similar enzymes suggests a double displacement reaction, while computer modelling favours front side SNi attack. The first mechanism requires Gln189 to act as a nucleophile at the anomeric carbon, forming an enzyme-substrate imidic ester intermediate which is then attacked by the 4-OH group of the acceptor sugar. Inferred proton transfer to the UDP leaving group phosphate. Gln189 has subsequently been shown to be a weak nucleophile where a SNi mechanism is far more favoured with other enzymes also being shown to follow the SNi path.

Catalytic Residues Roles

UniProt PDB* (1ga8)
Gln189 Gln189A Experimental evidence has suggested that Gln189 uses its side chain nitrogen as a nucleophile at the anomeric carbon of the alpha-D-galactose substrate. The enzyme-substrate imidic ester intermediate is then attacked by the 4-OH of the acceptor lactose, forming the product with retention of stereochemistry. However, no intermediate has been observed for this enzyme. covalently attached, nucleofuge, nucleophile
Asp130, Asp188 Asp130A, Asp188A The residue's side chain stabilises the transition state. electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

bimolecular nucleophilic substitution, enzyme-substrate complex formation, intermediate formation, overall reactant used, proton transfer, enzyme-substrate complex cleavage, overall product formed, intermediate collapse, inferred reaction step, native state of enzyme regenerated

References

  1. Persson K et al. (2001), Nat Struct Biol, 8, 166-175. Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs. DOI:10.1038/84168. PMID:11175908.
  2. Tvaroska I (2004), Carbohydr Res, 339, 1007-1014. Molecular modeling insights into the catalytic mechanism of the retaining galactosyltransferase LgtC. DOI:10.1016/j.carres.2003.11.014. PMID:15010308.

Step 1. Gln189 carbonyl acts as a nucleophile and attacks C1 on UDP-galactose to form a covalent intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gln189A covalently attached
Asp188A electrostatic stabiliser
Asp130A electrostatic stabiliser
Asp103A metal ligand
Asp105A metal ligand
His244A metal ligand
Gln189A nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, enzyme-substrate complex formation, intermediate formation, overall reactant used

Step 2. Nucleophilic attack of the 4'OH on the enzyme-substrate intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gln189A covalently attached
Asp103A metal ligand
Asp105A metal ligand
His244A metal ligand
Gln189A nucleofuge

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, enzyme-substrate complex cleavage, overall product formed, intermediate collapse, inferred reaction step, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Gln189 carbonyl acts as a nucleophile and attacks C1 on UDP-galactose to form a covalent intermediate.

Step 2. Nucleophilic attack of the 4'OH on the enzyme-substrate intermediate.

Products.

Contributors

James W. Murray, Craig Porter, Gemma L. Holliday, Morwenna Hall