2,6-dioxo-6-phenylhexa-3-enoate hydrolase

 

2-Hydroxyl-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase (BphD) hydrolyses an unusual C-C bond of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to produce benzoic acid and 2-hydroxy-2,4-pentadienoic acid (HPD). HOPDA is an aromatic compound generated in a biphenyl/polychlorinated biphenyl (PCB) degradation pathway of bacteria.

It is a member of the alpha/beta hydrolase superfamily and has the canonical Ser-His-Asp triad. There has been a lot of debate as to the exact mechanism of this enzyme.

 

Reference Protein and Structure

Sequence
Q75WN8 UniProt (3.7.1.8) IPR000073 (Sequence Homologues) (PDB Homologues)
Biological species
Rhodococcus jostii RHA1 (Bacteria) Uniprot
PDB
1c4x - 2-HYDROXY-6-OXO-6-PHENYLHEXA-2,4-DIENOATE HYDROLASE (BPHD) FROM RHODOCOCCUS SP. STRAIN RHA1 (2.4 Å) PDBe PDBsum 1c4x
Catalytic CATH Domains
3.40.50.1820 CATHdb (see all for 1c4x)
Click To Show Structure

Enzyme Reaction (EC:3.7.1.8)

2,6-dioxo-6-phenylhexa-3-enoate(1-)
CHEBI:64675ChEBI
+
water
CHEBI:15377ChEBI
→
hydron
CHEBI:15378ChEBI
+
benzoate
CHEBI:16150ChEBI
+
2-oxopent-4-enoate
CHEBI:11641ChEBI
Alternative enzyme names: HOHPDA hydrolase,

Enzyme Mechanism

Introduction

The exact mechanism is currently unclear and two proposals have been put forward.

The first (shown here) suggests that the Ser-His-Asp triad of the alpha/beta hydrolase fold superfamily members performs in the traditional way, with a covalent intermediate being formed between the serine and substrate.

In this mechanism, the free enzyme (E) binds and the 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid substrate undergoes keto-enol tautomerisation (via His263-catalysed transfer of a proton to the proS position at C-5), generating the first intermediate. Nucleophilic attack of Ser110 on this intermediate generates the first tetrahedral (covalently bound) intermediate, the negative charge of which is stabilised by the main chain amide protons of the 'oxyanion hole' residues, Met111 and Gly40. Collapse of this intermediate involves C-C fragmentation onto the re face of the double bond, generating the acyl-enzyme intermediate, and releasing 2-hydroxy-penta-2,4-dienoic acid with the inserted proton at the H-5E position. His263 activates water to attack at the acyl-enzyme carbonyl, generating the second tetrahedral intermediate, collapse of which releases benzoate and regenerates free enzyme.

Catalytic Residues Roles

UniProt PDB* (1c4x)
Asn110, Trp265 Asn109(110)A, Trp264(265)A Acts as an electrostatic stabiliser, activating the hydroxyl group for the initial tautomerisation reaction. electrostatic stabiliser
Asp236 Asp235(236)A Activates and stabilises the histidine general acid/base. modifies pKa
Ser111 Ser110(111)A Acts as a catalytic nucleophile. covalent catalysis, proton shuttle (general acid/base)
His264 His263(264)A Acts as a general acid/base. proton shuttle (general acid/base)
Gly41 (main-N), Met112 (main-N) Gly40(41)A (main-N), Met111(112)A (main-N) Forms the oxyanion hole. electrostatic stabiliser
Arg187 Arg186(187)A Responsible for binding the substrate dienol in a twisted, nonplanar conformation. This increases its reactivity toward ketonization. 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. Rauwerdink A et al. (2015), ACS Catal, 5, 6153-6176. How the Same Core Catalytic Machinery Catalyzes 17 Different Reactions: the Serine-Histidine-Aspartate Catalytic Triad of α/β-Hydrolase Fold Enzymes. DOI:10.1021/acscatal.5b01539.
  2. Li Y et al. (2015), RSC Adv, 5, 66591-66597. Insight into the catalytic mechanism of meta-cleavage product hydrolase BphD: a quantum mechanics/molecular mechanics study. DOI:10.1039/c5ra09939k.
  3. Ruzzini AC et al. (2013), Biochemistry, 52, 7428-7438. A Substrate-Assisted Mechanism of Nucleophile Activation in a Ser–His–Asp Containing C–C Bond Hydrolase. DOI:10.1021/bi401156a. PMID:24067021.
  4. Ruzzini AC et al. (2012), Biochemistry, 51, 5831-5840. The Catalytic Serine ofmeta-Cleavage Product Hydrolases Is Activated Differently for C–O Bond Cleavage Than for C–C Bond Cleavage. DOI:10.1021/bi300663r. PMID:22747426.
  5. Ruzzini AC et al. (2012), J Am Chem Soc, 134, 4615-4624. Identification of an Acyl-Enzyme Intermediate in ameta-Cleavage Product Hydrolase Reveals the Versatility of the Catalytic Triad. DOI:10.1021/ja208544g. PMID:22339283.
  6. Bhowmik S et al. (2007), J Biol Chem, 282, 36377-36385. The Molecular Basis for Inhibition of BphD, a C-C Bond Hydrolase Involved in Polychlorinated Biphenyls Degradation: LARGE 3-SUBSTITUENTS PREVENT TAUTOMERIZATION. DOI:10.1074/jbc.m707035200. PMID:17932031.
  7. Horsman GP et al. (2007), J Biol Chem, 282, 19894-19904. The Tautomeric Half-reaction of BphD, a C-C Bond Hydrolase: KINETIC AND STRUCTURAL EVIDENCE SUPPORTING A KEY ROLE FOR HISTIDINE 265 OF THE CATALYTIC TRIAD. DOI:10.1074/jbc.m702237200. PMID:17442675.
  8. Horsman GP et al. (2006), Biochemistry, 45, 11071-11086. Kinetic and Structural Insight into the Mechanism of BphD, a C−C Bond Hydrolase from the Biphenyl Degradation Pathway†. DOI:10.1021/bi0611098. PMID:16964968.
  9. Nandhagopal N et al. (2001), J Mol Biol, 309, 1139-1151. Crystal structure of 2-hydroxyl-6-oxo-6-phenylhexa-2,4-dienoic acid (HPDA) hydrolase (BphD enzyme) from the Rhodococcus sp. strain RHA1 of the PCB degradation pathway. DOI:10.1006/jmbi.2001.4737. PMID:11399084.

Step 1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly40(41)A (main-N) electrostatic stabiliser
Met111(112)A (main-N) electrostatic stabiliser
Ser110(111)A covalent catalysis, proton shuttle (general acid/base)
His263(264)A proton shuttle (general acid/base)
Trp264(265)A electrostatic stabiliser
Asn109(110)A electrostatic stabiliser
Arg186(187)A steric role
Asp235(236)A modifies pKa

Chemical Components

Step 1.

Introduction

The exact mechanism is currently unclear and two proposals have been put forward. This second mechanism proposal involves the formation of a gem-diolate intermediate. In this mechanism, the histidine of the Ser-His-Asp triad acts as a general acid/base and the serine acts to stabilise the reactive intermediates.

In this mechanism, the substrate is bound as the dienol form and deprotonated initially by His263. After a fast ketonization step, the C-6 ketone is twisted towards Ser110 and positioned for the nucleophilic attack of the catalytic water molecule, which is deprotonated by His263 and positioned by Ser110 through a strong hydrogen bond.

Catalytic Residues Roles

UniProt PDB* (1c4x)
Asn110, Trp265 Asn109(110)A, Trp264(265)A Acts as an electrostatic stabiliser, activating the hydroxyl group for the initial tautomerisation reaction. electrostatic stabiliser
Asp236 Asp235(236)A Activates the general acid/base histidine (His263). modifies pKa
Ser111 Ser110(111)A Has an important role in stabilising the oxyanion intermediate that is formed. electrostatic stabiliser
His264 His263(264)A Acts as a general acid/base. proton shuttle (general acid/base)
Gly41 (main-N), Met112 (main-N) Gly40(41)A (main-N), Met111(112)A (main-N) Forms the oxyanion hole. electrostatic stabiliser
Arg187 Arg186(187)A Arg188 is responsible for binding the substrate dienol in a twisted, nonplanar conformation. This increases its reactivity toward ketonization. electrostatic destabiliser
*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. Li JJ et al. (2007), Org Biomol Chem, 5, 507-513. Investigation of a general base mechanism for esterhydrolysis in C–C hydrolase enzymes of the α/β-hydrolase superfamily: a novel mechanism for the serine catalytic triad. DOI:10.1039/b615605c. PMID:17252134.
  2. Li C et al. (2006), Biochemistry, 45, 12470-12479. Catalytic Role for Arginine 188 in the C−C Hydrolase Catalytic Mechanism forEscherichia coliMhpC andBurkholderia xenovoransLB400 BphD†. DOI:10.1021/bi061253t. PMID:17029402.
  3. Li JJ et al. (2006), Biochemistry, 45, 12461-12469. Evidence for agem-Diol Reaction Intermediate in Bacterial C−C Hydrolase Enzymes BphD and MhpC from13C NMR Spectroscopy†. DOI:10.1021/bi0612519. PMID:17029401.
  4. Li C et al. (2005), J Mol Biol, 346, 241-251. Catalytic Mechanism of C–C Hydrolase MhpC from Escherichia coli: Kinetic Analysis of His263 and Ser110 Site-directed Mutants. DOI:10.1016/j.jmb.2004.11.032. PMID:15663941.
  5. Dunn G et al. (2005), J Mol Biol, 346, 253-265. The Structure of the C–C Bond Hydrolase MhpC Provides Insights into its Catalytic Mechanism. DOI:10.1016/j.jmb.2004.11.033. PMID:15663942.
  6. Speare DM et al. (2004), Org Biomol Chem, 2, 2942-2950. Synthetic 6-aryl-2-hydroxy-6-ketohexa-2,4-dienoic acid substrates for C–C hydrolase BphD: investigation of a general base catalytic mechanism. DOI:10.1039/b410322j. PMID:15480459.
  7. Fleming SM et al. (2000), Biochemistry, 39, 1522-1531. Catalytic Mechanism of a C−C Hydrolase Enzyme: Evidence for aGem-Diol Intermediate, Not an Acyl Enzyme†. DOI:10.1021/bi9923095.

Step 1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His263(264)A proton shuttle (general acid/base)
Ser110(111)A electrostatic stabiliser
Arg186(187)A electrostatic destabiliser
Asp235(236)A modifies pKa
Gly40(41)A (main-N) electrostatic stabiliser
Asn109(110)A electrostatic stabiliser
Met111(112)A (main-N) electrostatic stabiliser
Trp264(265)A electrostatic stabiliser

Chemical Components

Step 1.

Contributors

Nozomi Nagano, Craig Porter, Gemma L. Holliday