Myristoyl-ACP-specific thioesterase

 

Myristoyl acyl carrier protein specific thioesterase (C14ACP-TE) is a lux-specific acyltransferase belonging to the alpha/beta hydrolase family. The acyl transferase is part of the fatty acid reductase system required for aldehyde biosynthesis. It produces fatty acids for the light-emitting reaction catalysed by luciferase in luminescent bacteria. In Vibrio harveyi, C14ACP-TE is responsible for catalysing the transfer or hydrolysis of acyl group from either myristoyl-ACP or myristoyl-CoA to form myristic acid, and for diverting the myristic acid to the bioluminescence pathway, where it undergoes NADPH-dependent reduction and subsequent FMNH2- and O2-dependent oxidation of the corresponding aldehyde, with accompanying emission of light.

 

Reference Protein and Structure

Sequence
P05521 UniProt (2.3.1.-) IPR003157 (Sequence Homologues) (PDB Homologues)
Biological species
Vibrio harveyi (Bacteria) Uniprot
PDB
1tht - STRUCTURE OF A MYRISTOYL-ACP-SPECIFIC THIOESTERASE FROM VIBRIO HARVEYI (2.1 Å) PDBe PDBsum 1tht
Catalytic CATH Domains
3.40.50.1820 CATHdb (see all for 1tht)
Click To Show Structure

Enzyme Reaction (EC:2.3.1.-)

myristoyl-CoA(4-)
CHEBI:57385ChEBI
+
acyl-carrier protein
CHEBI:13534ChEBI
→
myristoyl-[acyl-carrier protein]
CHEBI:50651ChEBI
+
coenzyme A(4-)
CHEBI:57287ChEBI

Enzyme Mechanism

Introduction

Enzymatic hydrolysis of the thioester bond proceeds using the catalytic triad of Ser 114, Asp 211, and His 241. His 241 activates Ser 114 as a nucleophile by abstracting a proton from the serine hydroxyl as the carbonyl carbon of the substrate is attacked by the nucleophile. His 241 is stabilised by hydrogen bonding to Asp 211 which lowers the pKa of His so that it more willingly accepts a proton. This results in the formation of a tetrahedral transition state which collapses, with the His 241 donating a proton to the first leaving group (CoA), to form the acylenzyme intermediate. The intermediate is subsequently attacked by the activated ACP sulphur as it is deprotonated by His 241 to activate it. This second tetrahedral transition state will collapse resulting in the cleavage of the acyl-enzyme bond and the released Ser 114 will accept a proton from His 241 which regenerates the native state of the active site.

Catalytic Residues Roles

UniProt PDB* (1tht)
Asp211 Asp211A Stabilises His 241 by hydrogen bonding which as a result lowers His 241's pKa and so will more willingly accept a proton from Ser 114. modifies pKa, electrostatic stabiliser
Ser114 Ser114A Ser114 is activated by His241. It acts as a nucleophile and attacks the substrate to form the acylenzyme intermediate. Its backbone amide may also be involved in the formation of an oxyanion hole. covalently attached, nucleofuge, nucleophile, proton acceptor, proton donor
His241 His241A His241 acts as a general acid/base catalyst, activating Ser114 and water as nucleophiles. proton acceptor, proton donor
*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, intermediate formation, overall reactant used, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed, enzyme-substrate complex cleavage, native state of enzyme regenerated

References

  1. Li J et al. (1996), Biochemistry, 35, 9967-9973. Conversion of Serine-114 to Cysteine-114 and the Role of the Active Site Nucleophile in Acyl Transfer by Myristoyl-ACP Thioesterase fromVibrio harveyi†. DOI:10.1021/bi9605292. PMID:8756458.
  2. Lawson DM et al. (1994), Biochemistry, 33, 9382-9388. Structure of a Myristoyl-ACP-Specific Thioesterase from Vibrio harveyi. DOI:10.1021/bi00198a003. PMID:8068614.
  3. Ferri SR et al. (1994), J Biol Chem, 269, 6683-6688. An essential histidine residue required for fatty acylation and acyl transfer by myristoyltransferase from luminescent bacteria. PMID:8120025.

Step 1. His241 abstracts a proton from Ser114 and activates it for nucleophilic attack on the thioester carbonyl of myristoyl-CoA.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser114A covalently attached
Asp211A electrostatic stabiliser, modifies pKa
His241A proton acceptor
Ser114A proton donor, nucleophile

Chemical Components

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

Step 2. His241 protonates the sulphur that bridges the CoA extremity and myristoyl which results in the collapse of the tetrahedral intermediate and the cleavage of the S-C bond.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp211A electrostatic stabiliser, modifies pKa
Ser114A covalently attached
His241A proton donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, intermediate collapse, overall product formed

Step 3. His241 deprotonates the the sulphur of ACP which activates it to nucleophilically attack the carbonyl carbon of the acyl-enzyme intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp211A electrostatic stabiliser, modifies pKa
Ser114A covalently attached
His241A proton acceptor

Chemical Components

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

Step 4. The tetrahedral intermediate collapses resulting in the cleavage of the acyl-enzyme bond which releases Ser 114 which can now accept a proton from His 241.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp211A electrostatic stabiliser, modifies pKa
Ser114A covalently attached
His241A proton donor
Ser114A nucleofuge, proton acceptor

Chemical Components

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

Step 1. His241 abstracts a proton from Ser114 and activates it for nucleophilic attack on the thioester carbonyl of myristoyl-CoA.

Step 2. His241 protonates the sulphur that bridges the CoA extremity and myristoyl which results in the collapse of the tetrahedral intermediate and the cleavage of the S-C bond.

Step 3. His241 deprotonates the the sulphur of ACP which activates it to nucleophilically attack the carbonyl carbon of the acyl-enzyme intermediate.

Step 4. The tetrahedral intermediate collapses resulting in the cleavage of the acyl-enzyme bond which releases Ser 114 which can now accept a proton from His 241.

Products.

Contributors

Gemma L. Holliday, Charity Hornby