Thiaminase (class 1)

 

Thiaminase metabolises thiamine into two constituent parts, the pyrimidine unit, with the addition of a nucleophile, and hemineurine. In fern plants thiaminase is thought to provide protection from insect infestation, while in microorganisms and fish, in which the enzyme has also been found there is, as of yet no defined physiological role.

Two classes of thiaminase exist, class 1 (EC:2.5.1.2) and class 2 (EC:3.5.99.2). The two classes share no sequence homology, although their catalytic mechanisms are thought to be very similar, utilising the same active site residues. One difference is their substrate specificity: class 1 thiaminases will catalyse the incorporation of several nucleophiles into the departing pyridimium moiety, while class 2 thiaminases will only utilise water as a nucleophile. The substrate divergence is thought to stem from the lack of homology in protein sequence.

 

Reference Protein and Structure

Sequence
P45741 UniProt (2.5.1.2) IPR030901 (Sequence Homologues) (PDB Homologues)
Biological species
Paenibacillus thiaminolyticus (Bacteria) Uniprot
PDB
3thi - THIAMINASE I FROM BACILLUS THIAMINOLYTICUS (2.0 Å) PDBe PDBsum 3thi
Catalytic CATH Domains
3.40.190.10 CATHdb (see all for 3thi)
Click To Show Structure

Enzyme Reaction (EC:2.5.1.2)

thiamine(1+)
CHEBI:18385ChEBI
+
pyridine
CHEBI:16227ChEBI
→
1-[(4-amino-2-methylpyrimidin-5-yl)methyl]pyridinium
CHEBI:11222ChEBI
+
5-(2-hydroxyethyl)-4-methylthiazole
CHEBI:17957ChEBI
Alternative enzyme names: Pyrimidine transferase, Thiamine hydrolase, Thiamine pyridinolase, Thiamine pyridinylase, Thiamine:base 2-methyl-4-aminopyrimidine-5-methenyltransferase, Thiaminase, Thiaminase I,

Enzyme Mechanism

Introduction

Cys113 undergoes nucleophilic attack on the pyrimidine subunit, forming a zwitter-ionic adduct. Expulsion of the hemineurine leaving group via an Ec1b mechanism generates an alkene which is attacked by the incoming nucleophile, in this case pyridine, to form a new carbon-nitrogen bond.

Catalytic Residues Roles

UniProt PDB* (3thi)
Asp302 Asp272(264)A Asp272 is proposed to hydrogen bond with the pyrimidine ring, stabilising the substrate in a linear alignment with the Cys113 nucleophile. hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser, steric role
Glu271 Glu241(233)A Acts as a general acid/base, activating the nucleophilic cysteine residue. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, activator
Cys143 Cys113(105)A Acts as a nucleophile. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleophile, nucleofuge, proton acceptor, proton donor, activator
*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

proton transfer, bimolecular nucleophilic addition, enzyme-substrate complex formation, overall reactant used, intermediate formation, unimolecular elimination by the conjugate base, overall product formed, intermediate collapse, enzyme-substrate complex cleavage, native state of enzyme regenerated

References

  1. Costello CA et al. (1996), J Biol Chem, 271, 3445-3452. Mechanistic studies on thiaminase I. Overexpression and identification of the active site nucleophile. PMID:8631946.
  2. Kreinbring CA et al. (2014), Proc Natl Acad Sci U S A, 111, 137-142. Structure of a eukaryotic thiaminase I. DOI:10.1073/pnas.1315882110. PMID:24351929.
  3. Jenkins AL et al. (2008), Bioorg Chem, 36, 29-32. Mutagenesis studies on TenA: A thiamin salvage enzyme from Bacillus subtilis. DOI:10.1016/j.bioorg.2007.10.005. PMID:18054064.
  4. Campobasso N et al. (1998), Biochemistry, 37, 15981-15989. Crystal Structure of Thiaminase-I fromBacillusthiaminolyticusat 2.0 Å Resolution†,‡. DOI:10.1021/bi981673l. PMID:9843405.

Step 1. Glu241 deprotonates Cys113, activating this residue towards nucleophilic attack at the thiamine substrate, forming an covalently-bound adduct. The negative charge is stabilised as a zwitterion, with the accompanying positive charge residing on the thiazole nitrogen.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp272(264)A hydrogen bond donor, electrostatic stabiliser, steric role
Cys113(105)A activator, hydrogen bond donor
Glu241(233)A activator, hydrogen bond acceptor
Cys113(105)A nucleophile
Glu241(233)A proton acceptor
Cys113(105)A proton donor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, overall reactant used, intermediate formation

Step 2. The zwitterion intermediate undergoes intramolecular elimination, releasing hemineurine (5-2-hydroxyethyl-4-methylthiazole) and the enzyme-bound, enol intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp272(264)A hydrogen bond acceptor
Cys113(105)A activator, covalently attached
Glu241(233)A hydrogen bond donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, overall product formed, intermediate formation

Step 3. Pyridine attacks the enol intermediate. The resulting carbanion is stabilised as a zwitterion with the positive charge on the pyridine nitrogen.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp272(264)A hydrogen bond acceptor
Cys113(105)A activator, covalently attached
Glu241(233)A hydrogen bond donor

Chemical Components

ingold: bimolecular nucleophilic addition, overall reactant used, intermediate formation

Step 4. The zwitterion intermediate undergoes intramolecular elimination to release Cys113 and also the heteropyrithiamine product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp272(264)A hydrogen bond acceptor
Cys113(105)A activator, hydrogen bond acceptor
Glu241(233)A activator, hydrogen bond donor, proton donor
Cys113(105)A nucleofuge, proton acceptor

Chemical Components

ingold: unimolecular elimination by the conjugate base, overall product formed, intermediate collapse, enzyme-substrate complex cleavage, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Glu241 deprotonates Cys113, activating this residue towards nucleophilic attack at the thiamine substrate, forming an covalently-bound adduct. The negative charge is stabilised as a zwitterion, with the accompanying positive charge residing on the thiazole nitrogen.

Step 2. The zwitterion intermediate undergoes intramolecular elimination, releasing hemineurine (5-2-hydroxyethyl-4-methylthiazole) and the enzyme-bound, enol intermediate.

Step 3. Pyridine attacks the enol intermediate. The resulting carbanion is stabilised as a zwitterion with the positive charge on the pyridine nitrogen.

Step 4. The zwitterion intermediate undergoes intramolecular elimination to release Cys113 and also the heteropyrithiamine product.

Products.

Contributors

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