(S)-2-haloacid dehalogenase

 

(S)-2-haloacid dehalogenase catalyses the hydrolytic dehalogenation of L-2-haloacid to the corresponding D-2-hydroxyacid with the inversion of the configuration at the C2 atom. The degradation of halogenated hydrocarbons used in large quantities to make solvents and plastics. The stereospecificity of this enzyme is of interest for its potential use in the biosynthesis of chiral compounds. It is a member of the haloacid dehalogenase superfamily.

 

Reference Protein and Structure

Sequence
Q60099 UniProt (3.8.1.2) IPR006439 (Sequence Homologues) (PDB Homologues)
Biological species
Xanthobacter autotrophicus (Bacteria) Uniprot
PDB
1qq5 - STRUCTURE OF L-2-HALOACID DEHALOGENASE FROM XANTHOBACTER AUTOTROPHICUS (1.52 Å) PDBe PDBsum 1qq5
Catalytic CATH Domains
3.40.50.1000 CATHdb 1.10.150.240 CATHdb (see all for 1qq5)
Cofactors
Water (1)
Click To Show Structure

Enzyme Reaction (EC:3.8.1.2)

water
CHEBI:15377ChEBI
+
(S)-2-chloropropanoate
CHEBI:73934ChEBI
→
chloride
CHEBI:17996ChEBI
+
(R)-lactate
CHEBI:16004ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: 2-haloacid dehalogenase, 2-haloacid halidohydrolase, 2-haloalkanoic acid dehalogenase, 2-haloalkanoid acid halidohydrolase, 2-halocarboxylic acid dehalogenase II, DL-2-haloacid dehalogenase, L-2-haloacid dehalogenase, L-DEX, Halocarboxylic acid halidohydrolase,

Enzyme Mechanism

Introduction

When the L-2-haloacid is taken into the active site its carboxyl group is recognised by Arg41 before forming a Michaelis compound with the enzyme. The substrate binding drives out a water molecule freeing Asp10's nucleophilic oxygen allowing attack on the C2 carbon via an SN2 mechanism. Asp10 is stabilised by hydrogen bonding to Lys151, Thr14 and Ser175. Arg41 is thought to abstract the halide ion from the substrate. Ser118 serves as the main residue for stabilising the substrate carboxyl moiety in the reaction intermediate. Asn177 and Asp180 are also thought to be involved in the hydrolysis reaction.

Catalytic Residues Roles

UniProt PDB* (1qq5)
Asp8 Asp8A Acts as the catalytic nucleophile, forming a covalently bound intermediate with the substrate. hydrogen bond acceptor, nucleofuge, nucleophile
Asp176 Asp176A Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, activator
Arg39, Phe175, Asn115 Arg39A, Phe175A, Asn115A Stabilise the chloride leaving group. hydrogen bond donor, electrostatic stabiliser
Lys147, Thr12, Ser171, Asn173, Ser114 Lys147A, Thr12A, Ser171A, Asn173A, Ser114A Activate the substrate. activator, hydrogen bond 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

bimolecular nucleophilic substitution, atom stereo change, overall reactant used, overall product formed, enzyme-substrate complex formation, intermediate formation, proton transfer, enzyme-substrate complex cleavage, intermediate terminated, intermediate collapse, hydrolysis, native state of enzyme regenerated, inferred reaction step

References

  1. Li YF et al. (1998), J Biol Chem, 273, 15035-15044. Crystal Structures of Reaction Intermediates ofL-2-Haloacid Dehalogenase and Implications for the Reaction Mechanism. DOI:10.1074/jbc.273.24.15035. PMID:9614112.
  2. Kumar A et al. (2016), Int J Biol Macromol, 83, 216-225. l-2-Haloacid dehalogenase from Ancylobacter aquaticus UV5: Sequence determination and structure prediction. DOI:10.1016/j.ijbiomac.2015.11.066. PMID:26645146.
  3. Paneth P (2003), Acc Chem Res, 36, 120-126. Chlorine Kinetic Isotope Effects on Enzymatic Dehalogenations. DOI:10.1021/ar010101h. PMID:12589697.
  4. Lahiri SD et al. (2002), Biochemistry, 41, 8351-8359. Caught in the Act:  The Structure of Phosphorylatedβ-Phosphoglucomutase fromLactococcus lactis†,‡. DOI:10.1021/bi0202373.
  5. Ichiyama S et al. (2000), J Biol Chem, 275, 40804-40809. Novel Catalytic Mechanism of Nucleophilic Substitution by Asparagine Residue Involving Cyanoalanine Intermediate Revealed by Mass Spectrometric Monitoring of an Enzyme Reaction. DOI:10.1074/jbc.m008065200. PMID:11006296.
  6. Morais MC et al. (2000), Biochemistry, 39, 10385-10396. The Crystal Structure ofBacillus cereusPhosphonoacetaldehyde Hydrolase:  Insight into Catalysis of Phosphorus Bond Cleavage and Catalytic Diversification within the HAD Enzyme Superfamily†,‡. DOI:10.1021/bi001171j.
  7. Ridder IS et al. (1999), J Biol Chem, 274, 30672-30678. Crystal Structures of Intermediates in the Dehalogenation of Haloalkanoates by L-2-Haloacid Dehalogenase. DOI:10.1074/jbc.274.43.30672. PMID:10521454.
  8. Kurihara T et al. (1995), J Biochem, 117, 1317-1322. Comprehensive site-directed mutagenesis of L-2-halo acid dehalogenase to probe catalytic amino acid residues. PMID:7490277.

Step 1. Asp8 initiates a nucleophilic attack upon the chlorinated carbon of the substrate in a substitution reaction, eliminating chloride.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp176A hydrogen bond acceptor, activator
Arg39A hydrogen bond donor
Lys147A hydrogen bond donor, activator
Ser171A hydrogen bond donor, electrostatic stabiliser
Thr12A hydrogen bond donor, electrostatic stabiliser
Asn173A hydrogen bond donor, electrostatic stabiliser
Asp8A hydrogen bond acceptor
Phe175A polar/non-polar interaction
Asn115A hydrogen bond donor
Ser114A electrostatic stabiliser
Asp8A nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, atom stereo change, overall reactant used, overall product formed, enzyme-substrate complex formation, intermediate formation

Step 2. Asp176 deprotonates water, which initiates a nucleophilic attack on the carbonyl of Asp8, as demonstrated by isotope studies, eliminating Asp8 while maintaining R stereochemistry, which is inverted to that of the S-haloacid substrate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp176A hydrogen bond acceptor
Arg39A hydrogen bond donor, electrostatic stabiliser
Lys147A hydrogen bond donor
Ser171A hydrogen bond donor, electrostatic stabiliser
Asn115A hydrogen bond donor, hydrogen bond acceptor, electrostatic stabiliser
Thr12A hydrogen bond donor, electrostatic stabiliser
Asn173A hydrogen bond donor, electrostatic stabiliser
Asp8A hydrogen bond acceptor
Phe175A polar/non-polar interaction, electrostatic stabiliser
Ser114A electrostatic stabiliser
Asp8A nucleofuge
Asp176A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, overall product formed, enzyme-substrate complex cleavage, intermediate terminated, intermediate collapse, hydrolysis

Step 3. In an inferred return step, water deprotonates Asp176.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp176A hydrogen bond donor
Lys147A hydrogen bond donor
Ser171A hydrogen bond donor, electrostatic stabiliser
Thr12A hydrogen bond donor, electrostatic stabiliser
Asn173A hydrogen bond donor, electrostatic stabiliser
Asp8A hydrogen bond acceptor
Asp176A proton donor

Chemical Components

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

Step 1. Asp8 initiates a nucleophilic attack upon the chlorinated carbon of the substrate in a substitution reaction, eliminating chloride.

Step 2. Asp176 deprotonates water, which initiates a nucleophilic attack on the carbonyl of Asp8, as demonstrated by isotope studies, eliminating Asp8 while maintaining R stereochemistry, which is inverted to that of the S-haloacid substrate.

Step 3. In an inferred return step, water deprotonates Asp176.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Sophie T. Williams, Alex Gutteridge, Craig Porter