DNA beta-glucosyltransferase

 

DNA beta-glucosyltransferase (BGT) is an enzyme encoded by a number of bacteriophage belonging to the T-even group. It catalyses the transfer of glucose from uridine diphosphoglucose (UDP-glucose) to 5-hydroxymethylcytosine (5-HMC) bases in double stranded DNA. Such glucosylation protects the infecting viral DNA from host restrictive enzymes. It may, in addition, be involved in phage specific gene expression by influencing transcription. Glucosylation also occurs in Trypanosoma bruceii where it is thought to be involved in regulating the expression of variant surface glycoproteins used by the parasite for protection against immune recognition. BGT is one of two enzymes involved in the glucosylation. In contrast to its counterpart which catalyses the formation of alpha-, it forms beta-glycosidic linkages

While a divalent cation is required for optimal catalytic activity, crystallographic data suggests that the metal and substrate do not occupy the active site at the same time, and therefore, casts doubt on whether the metal is necessary for a non-reaction function, such as product displacement.

 

Reference Protein and Structure

Sequence
P04547 UniProt (2.4.1.27) IPR015281 (Sequence Homologues) (PDB Homologues)
Biological species
Enterobacteria phage T4 (Virus) Uniprot
PDB
1c3j - T4 PHAGE BETA-GLUCOSYLTRANSFERASE: SUBSTRATE BINDING AND PROPOSED CATALYTIC MECHANISM (1.88 Å) PDBe PDBsum 1c3j
Catalytic CATH Domains
3.40.50.2000 CATHdb (see all for 1c3j)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:2.4.1.27)

UDP-D-glucose(2-)
CHEBI:58367ChEBI
+
5-hydroxymethyldeoxycytidine 5'-monophosphate residue
CHEBI:137351ChEBI
→
5'-glucosyl-methylhydroxycytosine
CHEBI:137352ChEBI
+
hydron
CHEBI:15378ChEBI
+
UDP(3-)
CHEBI:58223ChEBI
Alternative enzyme names: T4 phage beta-glucosyltransferase, T(4)-beta-glucosyl transferase, T(4)-HMC-beta-glucosyl transferase, UDP glucose-DNA beta-glucosyltransferase, UDPglucose:DNA beta-D-glucosyltransferase, Uridine diphosphoglucose-deoxyribonucleate beta-glucosyltransferase, UDP-glucose:DNA beta-D-glucosyltransferase,

Enzyme Mechanism

Introduction

Although previous proposals for a mechanism involve a general acid catalysis requiring two critical residues, a proton donor and a nucleophile/base current models suggest an alternative. BGT binds two substrates, UDP-glucose and 5-HMC. Glucose transfer occurs by a direct nucleophilic attack by the hydroxyl group of 5-HMC to form a beta-glucosidic bond followed by inversion of configuration. A catalytic base, thought to be Asp100 or Glu22, is provided by the enzyme to abstract a proton from the hydroxyl group activating the attack. Multiple contacts are thought to be made by the enzyme on the substrates but as yet are all speculative.

Catalytic Residues Roles

UniProt PDB* (1c3j)
Arg191 Arg191A Enzyme activity has been associated with a domain rearrangement, thought to be mediated by the ionic interaction between Asp100 and Arg191. hydrogen bond donor, electrostatic stabiliser, steric role
Phe72 Phe72A Phe72 is thought to be important for intercalating into the DNA duplex and anchoring the substrate in place. steric role, electrostatic stabiliser
Asp100, Glu22 Asp100A, Glu22A General acid/base. There is some debate in the literature as to whether the base is Asp100 or Glu22. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, activator, electrostatic stabiliser, steric role
*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 reactant used, overall product formed, inferred reaction step, native state of enzyme regenerated

References

  1. Unligil UM et al. (2000), Curr Opin Struct Biol, 10, 510-517. Glycosyltransferase structure and mechanism. DOI:10.1016/s0959-440x(00)00124-x. PMID:11042447.
  2. Tvaroška I (2015), Carbohydr Res, 403, 38-47. Atomistic insight into the catalytic mechanism of glycosyltransferases by combined quantum mechanics/molecular mechanics (QM/MM) methods. DOI:10.1016/j.carres.2014.06.017. PMID:25060837.
  3. Lairson LL et al. (2008), Annu Rev Biochem, 77, 521-555. Glycosyltransferases: structures, functions, and mechanisms. DOI:10.1146/annurev.biochem.76.061005.092322. PMID:18518825.
  4. Larivière L et al. (2003), J Mol Biol, 330, 1077-1086. Crystal Structures of the T4 Phage β-Glucosyltransferase and the D100A Mutant in Complex with UDP-glucose: Glucose Binding and Identification of the Catalytic Base for a Direct Displacement Mechanism. DOI:10.1016/s0022-2836(03)00635-1. PMID:12860129.
  5. Moréra S et al. (1999), J Mol Biol, 292, 717-730. T4 Phage β-Glucosyltransferase: Substrate Binding and Proposed Catalytic Mechanism. DOI:10.1006/jmbi.1999.3094. PMID:10497034.
  6. Murray BW et al. (1997), Biochemistry, 36, 823-831. Mechanism of Human α-1,3-Fucosyltransferase V:  Glycosidic Cleavage Occurs Prior to Nucleophilic Attack†. DOI:10.1021/bi962284z. PMID:9020780.
  7. Davies G et al. (1995), Structure, 3, 853-859. Structures and mechanisms of glycosyl hydrolases. DOI:10.1016/s0969-2126(01)00220-9. PMID:8535779.

Step 1. BGT binds two substrates, UDP-glucose and 5-HMC. Glucose transfer occurs by a direct nucleophilic attack by the hydroxyl group of 5-HMC to form a beta-glycosidic bond followed by inversion of configuration. A catalytic base, Asp100, is provided by the enzyme to abstract a proton from the hydroxyl group activating the attack. Glucose transfer proceeds via an oxocarbenium ion-character transition state, resulting in an inversion of configuration and the formation of a beta-glycosidic linkage.

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Catalytic Residues Roles

Residue Roles
Asp100A activator, hydrogen bond acceptor, electrostatic stabiliser, steric role
Arg191A hydrogen bond donor, electrostatic stabiliser, steric role
Phe72A steric role, electrostatic stabiliser
Glu22A hydrogen bond acceptor
Asp100A proton acceptor

Chemical Components

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

Step 2. Asp100 is deprotonated and the active site is regenerated.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp100A activator, hydrogen bond donor
Arg191A hydrogen bond donor, electrostatic stabiliser
Asp100A proton donor

Chemical Components

proton transfer, inferred reaction step, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. BGT binds two substrates, UDP-glucose and 5-HMC. Glucose transfer occurs by a direct nucleophilic attack by the hydroxyl group of 5-HMC to form a beta-glycosidic bond followed by inversion of configuration. A catalytic base, Asp100, is provided by the enzyme to abstract a proton from the hydroxyl group activating the attack. Glucose transfer proceeds via an oxocarbenium ion-character transition state, resulting in an inversion of configuration and the formation of a beta-glycosidic linkage.

Step 2. Asp100 is deprotonated and the active site is regenerated.

Products.

Contributors

Sophie T. Williams, Nozomi Nagano, Craig Porter, Gemma L. Holliday, Charity Hornby