UDP-N-acetylmuramoylpentapeptide-lysine N6-alanyltransferase

 

FemX uses aminoacyl-tRNA as an amino acid donor to synthesise the peptide cross-bridge in peptidoglycan, apart of the cell wall of gram-positive bacteria. FemX belongs to the Fem family of nonribosomal peptidyl transferases . FemX is required for cell survival and is a target for beta-lactam antibiotics.

 

Reference Protein and Structure

Sequence
Q9EY50 UniProt (2.3.2.10) IPR003447 (Sequence Homologues) (PDB Homologues)
Biological species
Weissella viridescens (Bacteria) Uniprot
PDB
1p4n - Crystal Structure of Weissella viridescens FemX:UDP-MurNAc-pentapeptide complex (1.9 Å) PDBe PDBsum 1p4n
Catalytic CATH Domains
3.40.630.30 CATHdb (see all for 1p4n)
Click To Show Structure

Enzyme Reaction (EC:2.3.2.10)

3'-(L-alanyl)adenylyl zwitterionic group
CHEBI:78497ChEBI
+
UDP-N-acetyl-alpha-D-muramoyl-L-alanyl-gamma-D-glutamyl-L-lysyl-D-alanyl-D-alaninate(3-)
CHEBI:70758ChEBI
→
hydron
CHEBI:15378ChEBI
+
AMP 3'-end(1-) residue
CHEBI:78442ChEBI
+
UDP-N-acetylmuramoyl-L-alanyl-gamma-D-glutamyl-N(6)-(L-alanyl)-L-lysyl-D-alanyl-D-alaninate(3-)
CHEBI:71358ChEBI
Alternative enzyme names: UDP-N-acetylmuramoylpentapeptide lysine N(6)-alanyltransferase, Alanyl-transfer ribonucleate-uridine diphosphoacetylmuramoylpentapeptide transferase, Uridine diphosphoacetylmuramoylpentapeptide lysine N(6)-alanyltransferase, L-alanyl-tRNA:UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysyl-D-alanyl-D-alanine 6-N-alanyltransferase, L-alanyl-tRNA:UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysyl-D-alanyl-D-alanine N(6)-alanyltransferase,

Enzyme Mechanism

Introduction

The reaction is thought to proceed in a substrate-assisted manner, with no acid or base catalyst within range. Substrate-lysine performs a nucleophilic attack on the Ala-tRNA ester bond to form a negatively charged tetrahedral intermediate, stabilised by Lys306. The intermediate collapses from concomitant internal proton shuttle to form the products; tRNA and alanyl-UM5P, a peptidoglycan precursor. Phe305 is also conserved in Fem aminoacyl transferases, pi-stacking with the C75 ribose ring which when mutated significantly decreased enzymatic turnover.

Catalytic Residues Roles

UniProt PDB* (1p4n)
Lys306 Lys305A Hydrogen bond donor to the oxyanion intermediate, helping stabilise the negative charge on the carbonyl oxygen. hydrogen bond donor, electrostatic stabiliser
Phe305 Phe304A Catalytically conserved residue in Fem aminoacyl transferases that has pi-stacking interactions with the ribose sugar on tRNA. van der waals interaction, pi-pi interaction
*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

intermediate formation, bimolecular nucleophilic addition, overall reactant used, intermediate terminated, unimolecular elimination by the conjugate base, overall product formed, proton relay

References

  1. Fonvielle M et al. (2013), Angew Chem Int Ed Engl, 52, 7278-7281. The structure of FemX(Wv) in complex with a peptidyl-RNA conjugate: mechanism of aminoacyl transfer from Ala-tRNA(Ala) to peptidoglycan precursors. DOI:10.1002/anie.201301411. PMID:23744707.
  2. Moutiez M et al. (2017), Chem Rev, 117, 5578-5618. Aminoacyl-tRNA-Utilizing Enzymes in Natural Product Biosynthesis. DOI:10.1021/acs.chemrev.6b00523. PMID:28060488.
  3. Favrot L et al. (2016), Biochemistry, 55, 989-1002. Bacterial GCN5-Related N-Acetyltransferases: From Resistance to Regulation. DOI:10.1021/acs.biochem.5b01269. PMID:26818562.

Step 1. Amine on UDP-N-acetyl-muramyl-pentapeptide acts as a nucleophile and attacks the carbonyl carbon to form a tetrahedral intermediate. Lys306 stabilises the oxyanion intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys305A electrostatic stabiliser, hydrogen bond donor
Phe304A pi-pi interaction, van der waals interaction

Chemical Components

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

Step 2. Substrate assisted - proton shuttle resulting in collapse of the tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys305A electrostatic stabiliser
Glu319A hydrogen bond donor
Phe304A pi-pi interaction, van der waals interaction

Chemical Components

intermediate terminated, ingold: unimolecular elimination by the conjugate base, overall product formed, proton relay
Download: Image, Marvin File

Step 1. Amine on UDP-N-acetyl-muramyl-pentapeptide acts as a nucleophile and attacks the carbonyl carbon to form a tetrahedral intermediate. Lys306 stabilises the oxyanion intermediate.

Step 2. Substrate assisted - proton shuttle resulting in collapse of the tetrahedral intermediate.

Products.

Introduction

FemX catalyses the transfer of L-Ala onto the cytoplasmic precursor UDP-N-acetyl-muramyl-pentapeptide. Lys36 and Arg211 activate the substrate by maintaining it in a bent conformation. UDP-N-acetyl-muramyl-pentapeptide (UDP-MPP) is bound, followed by Ala-tRNA. Nucleophilic attack occurs with the lysine residue of UDP-MPP on the carbonyl of the aminoacylated-tRNA to generate a tetrahedral intermediate. Collapse of the tetrahedral intermediate is assisted by the action of Glu319 as a general acid to protonate the 3'-OH of the tRNA ribose moiety and Asp108 acting as a general base to abstract a proton from the amino group on UDP-MPP.

Catalytic Residues Roles

UniProt PDB* (1p4n)
Asp109 Asp108A Acts as a general base catalyst in activating the substrate for nucleophilic attack. proton acceptor
Lys37, Arg212 Lys36A, Arg211A Activates the substrate sterically. hydrogen bond donor, steric role
Glu320 Glu319A Acts as the general acid catalyst in protonation of the leaving group to facilitate cleavage. 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

overall reactant used, bimolecular nucleophilic addition, intermediate formation, unimolecular elimination by the conjugate base, intermediate terminated, proton transfer, overall product formed, inferred reaction step, native state of enzyme regenerated

References

  1. Hegde SS et al. (2003), J Biol Chem, 278, 22861-22867. Kinetic and Mechanistic Characterization of Recombinant Lactobacillus viridescens FemX (UDP-N-acetylmuramoyl Pentapeptide-lysine N6-Alanyltransferase). DOI:10.1074/jbc.m301565200. PMID:12679335.
  2. Fonvielle M et al. (2013), Angew Chem Int Ed Engl, 52, 7278-7281. The structure of FemX(Wv) in complex with a peptidyl-RNA conjugate: mechanism of aminoacyl transfer from Ala-tRNA(Ala) to peptidoglycan precursors. DOI:10.1002/anie.201301411. PMID:23744707.
  3. Maillard AP et al. (2005), J Bacteriol, 187, 3833-3838. Structure-Based Site-Directed Mutagenesis of the UDP-MurNAc-Pentapeptide-Binding Cavity of the FemX Alanyl Transferase from Weissella viridescens. DOI:10.1128/jb.187.11.3833-3838.2005. PMID:15901708.

Step 1. Amine on UDP-N-acetyl-muramyl-pentapeptide acts as a nucleophile and attacks the carbonyl carbon to form a tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys36A steric role
Arg211A steric role
Lys36A hydrogen bond donor
Arg211A hydrogen bond donor

Chemical Components

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

Step 2. Collapse of tetrahedral intermediate with Glu319 and Asp108 acting as a general acid and base to form the reaction products.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys36A hydrogen bond donor
Arg211A hydrogen bond donor
Lys36A steric role
Arg211A steric role
Asp108A proton acceptor
Glu319A proton donor

Chemical Components

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

Step 3. Inferred protonation step of Glu319.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu319A proton acceptor

Chemical Components

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

Step 1. Amine on UDP-N-acetyl-muramyl-pentapeptide acts as a nucleophile and attacks the carbonyl carbon to form a tetrahedral intermediate.

Step 2. Collapse of tetrahedral intermediate with Glu319 and Asp108 acting as a general acid and base to form the reaction products.

Step 3. Inferred protonation step of Glu319.

Products.

Contributors

Gary McDowell, Gemma L. Holliday, Morwenna Hall