PDBsum entry 5ejm

Transferase

PDB id: 5ejm

Contents

Protein chains

(+ 2 more) 556 a.a.

Ligands

TD5 ×8

GOL ×8

EDO ×8

Metals

_MN ×8

Waters ×4650

PDB id:

5ejm

Name:

Transferase

Title:

Thdp-mn2+ complex of r413a variant of ecmend soaked with 2- ketoglutarate for 35 min

Structure:

2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1- carboxylate synthase. Chain: a, b, c, d, e, f, g, h. Synonym: thdp-dependent enzyme mend,sephchc synthase,menaquinone biosynthesis protein mend. Engineered: yes. Mutation: yes

Source:

Escherichia coli k12. Organism_taxid: 83333. Strain: k12 substr. Mg1655. Gene: mend, b2264, jw5374. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

1.72Å R-factor: 0.165 R-free: 0.192

Authors:

H.G.Song,C.Dong,Y.Z.Chen,Y.R.Sun,Z.H.Guo

Key ref:

M.Qin et al. (2018). Two active site arginines are critical determinants of substrate binding and catalysis in MenD: a thiamine-dependent enzyme in menaquinone biosynthesis. Biochem J, 475, 3651-3667. PubMed id: 30341164 DOI: 10.1042/BCJ20180548

Date:

02-Nov-15 Release date: 02-Nov-16

Protein chains

P17109  (MEND_ECOLI) -  2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase from Escherichia coli (strain K12)

Seq: 556 a.a.

Struc: 556 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.2.2.1.9 - 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic-acid
• Reaction: isochorismate + 2-oxoglutarate + H⁺ = 5-enolpyruvoyl-6-hydroxy-2- succinyl-cyclohex-3-ene-1-carboxylate + CO2

isochorismate (Bound ligand (Het Group name = GOL) matches with 45.45% similarity) + 2-oxoglutarate + H(+) = 5-enolpyruvoyl-6-hydroxy-2- succinyl-cyclohex-3-ene-1-carboxylate + CO2 (Bound ligand (Het Group name = EDO) matches with 40.00% similarity)

• Cofactor: Mg(2+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1042/BCJ20180548

Biochem J 475:3651-3667 (2018)

PubMed id: 30341164

Two active site arginines are critical determinants of substrate binding and catalysis in MenD: a thiamine-dependent enzyme in menaquinone biosynthesis.

M.Qin, H.Song, X.Dai, Y.Chen, Z.Guo.

ABSTRACT

The bacterial enzyme MenD, or 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) synthase, catalyzes an essential Stetter reaction in menaquinone (vitamin K2) biosynthesis via thiamine diphosphate (ThDP)-bound tetrahedral post-decarboxylation intermediates. The detailed mechanism of this intermediate chemistry, however, is still poorly understood, but of significant interest given that menaquinone is an essential electron transporter in many pathogenic bacteria. Here, we used site-directed mutagenesis, enzyme kinetic assays, and protein crystallography to reveal an active-inactive intermediate equilibrium in MenD catalysis and its modulation by two conserved active site arginine residues. We observed that these conserved residues play a key role in shifting the equilibrium to the active intermediate by orienting the C₂-succinyl group of the intermediates through strong ionic hydrogen bonding. We found that when this interaction is moderately weakened by amino acid substitutions, the resulting proteins are catalytically competent with the C₂-succinyl group taking either the active or the inactive orientation in the post-decarboxylation intermediate. When this hydrogen-bonding interaction was strongly weakened, the succinyl group was re-oriented by 180° relative to the native intermediate, resulting in the reversal of the stereochemistry at the reaction center that disabled catalysis. Interestingly, this inactive intermediate was formed with a distinct kinetic behavior, likely as a result of a non-native mode of enzyme-substrate interaction. The mechanistic insights gained from these findings improve our understanding of the new ThDP-dependent catalysis. More importantly, the non-native-binding site of the inactive MenD intermediate uncovered here provides a new target for the development of antibiotics.