3-amino-5-hydroxybenzoate synthase

 

Catalyzes the dehydration and aromatization of 5-amino- 5-deoxy-3-dehydroshikimate (aminoDHS) to 3-amino-5-hydroxybenzoate (AHBA), a compound that then serves as the starter unit for the assembly of a polyketide during the biosynthesis of rifamycin B and other ansamycin antibiotics. Cannot utilize 5-deoxy-5-amino-3- dehydroquinate (aminoDHQ), 5-deoxy-5-aminoshikimate (aminoSA), quinate, 3-dehydroquinate, or 3-dehydroshikimate (DHS) as substrate.

In a complex with RifL, RifK may have a second function in the AHBA pathway, acting as a transaminase introducing the nitrogen into the first pathway intermediate, UDP-3-keto-D-glucose, to give UDP-kanosamine. Appears to use glutamine as the nitrogen donor; NH4+ or asparagine are 30% less effective as nitrogen donors and neither glutamate nor aspartate show activity.

 

Reference Protein and Structure

Sequence
O52552 UniProt (2.6.1.-, 4.2.1.144) IPR000653 (Sequence Homologues) (PDB Homologues)
Biological species
Amycolatopsis mediterranei S699 (Bacteria) Uniprot
PDB
1b9h - CRYSTAL STRUCTURE OF 3-AMINO-5-HYDROXYBENZOIC ACID (AHBA) SYNTHASE (2.0 Å) PDBe PDBsum 1b9h
Catalytic CATH Domains
3.40.640.10 CATHdb (see all for 1b9h)
Cofactors
Pyridoxal 5'-phosphate(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:4.2.1.144)

5-amino-5-deoxy-3-dehydroshikimic acid zwitterion
CHEBI:71963ChEBI
→
3-amino-5-hydroxybenzoate
CHEBI:71959ChEBI
+
water
CHEBI:15377ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: AHBA synthase, RifK (gene name),

Enzyme Mechanism

Introduction

The aromatization of aminoDHS to AHBA involves the formation of a Schiff base between the substrate and PLP (and eventually between Lys188 and PLP to eliminate the final product), and several proton transfer steps; that is, (1) abstraction of the C-5 proton, (2) protonation of 4-OH to make it a better leaving group, (3) protonation of the C-3 carbonyl group as part of the enolisation and (4) abstraction of the pro-6S proton. A modelled structure of AHBA synthase with bound substrate shows that the Lys-188 eta-amino group is positioned in proximity to 4-OH, H-5 and H-6S, and it is proposed that this residue mediates sequentially the steps (1), (2) and (4). However, it is too far removed from 3-O to catalyse step (3) and no other amino acid is close enough to catalyse this protonation directly. Instead, it is proposed that Q185 assists in the elimination of the protonated 4-OH as water and then the transfer of a proton from this water molecule to 3-O in a relay mechanism.

Catalytic Residues Roles

UniProt PDB* (1b9h)
Lys188 Lys188A The PLP cofactor in the holoenzyme forms a Schiff's base with K188. This residue also acts as a general acid base throughout the reaction. covalent catalysis, proton shuttle (general acid/base)
Asp159 Asp159A Forms a salt bridge with the pyridine nitrogen of the PLP cofactor. electrostatic stabiliser
Phe88 Phe88A Stabilises the enzyme–substrate complex by hydrophobic interactions with the ring of aminoDHS/AHBA electrostatic stabiliser
Gln185 Gln185A Acts as the second general acid/base required for the reaction to occur. proton shuttle (general acid/base)
His162, Arg219, Tyr226, Arg236 His162A, Arg219A(AA), Tyr226A(AA), Arg236A(AA) Involved in binding the PLP cofactor in the active site, maintaining its correct orientation and stabilising it. 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

References

  1. Floss HG et al. (2011), J Antibiot (Tokyo), 64, 35-44. The biosynthesis of 3-amino-5-hydroxybenzoic acid (AHBA), the precursor of mC7N units in ansamycin and mitomycin antibiotics: a review. DOI:10.1038/ja.2010.139. PMID:21081954.
  2. Eads JC et al. (1999), Biochemistry, 38, 9840-9849. Crystal Structure of 3-Amino-5-hydroxybenzoic Acid (AHBA) Synthase†,‡. DOI:10.1021/bi990018q. PMID:10433690.
  3. Kim CG et al. (1998), J Biol Chem, 273, 6030-6040. 3-Amino-5-hydroxybenzoic acid synthase, the terminal enzyme in the formation of the precursor of mC7N units in rifamycin and related antibiotics. PMID:9497318.

Step 1.

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Catalytic Residues Roles

Residue Roles
Lys188A covalent catalysis, proton shuttle (general acid/base)
Gln185A proton shuttle (general acid/base)
Asp159A electrostatic stabiliser
His162A steric role
Arg219A(AA) steric role
Tyr226A(AA) steric role
Arg236A(AA) steric role
Phe88A electrostatic stabiliser

Chemical Components

Step 1.

Contributors

Alex Gutteridge, Craig Porter, Gemma L. Holliday