Roles of K151 and D180 in L‐2‐haloacid dehalogenase from Pseudomonas sp. YL: Analysis by molecular dynamics and ab initio fragment molecular orbital calculations

Abstract

L-2-haloacid dehalogenase (L-DEX) catalyzes the hydrolytic dehalogenation of L-2-haloalkanoic acids to produce the corresponding D-2-hydroxyalkanoic acids. This enzyme is expected to be applicable to the bioremediation of environments contaminated with halogenated organic compounds. We analyzed the reaction mechanism of L-DEX from Pseudomonas sp. YL (L-DEX YL) by using molecular modeling. The complexes of wild-type L-DEX YL and its K151A and D180A mutants with its typical substrate, L-2-chloropropionate, were constructed by docking simulation. Subsequently, molecular dynamics (MD) and ab initio fragment molecular orbital (FMO) calculations of the complexes were performed. The ab initio FMO method was applied at the MP2/6-31G level to estimate interfragment interaction energies. K151 and D180, which are experimentally shown to be important for enzyme activity, interact particularly strongly with L-2-chloropropionate, catalytic water, nucleophile (D10), and with each other. Our calculations suggest that K151 stabilizes substrate orientation and balances the charge around the active site, while D180 stabilizes the rotation of the nucleophile D10, fixes catalytic water around D10, and prevents K151 from approaching D10. Further, D180 may activate catalytic water on its own or with K151, S175, and N177. These roles are consistent with the previous results. Thus, MD and ab initio FMO calculations are powerful tools for the elucidation of the mechanism of enzymatic reaction at the molecular level and can be applied to other catalytically important residues. The results obtained here will play an important role in elucidating the reaction mechanism and rational design of L-DEX YL with improved enzymatic activity or substrate specificity.

A Mechanistic Analysis of Enzymatic Degradation of Organohalogen Compounds

“…YL (L-DEX YL) is one of the best studied enzymes of the HAD superfamily. 6,[9][10][11][12][13][14][15][16][17][18][19][20][21][22] Mass spectrometry has played an important role in analyzing the reaction mechanism of L-DEX YL.…”

“…22) The complexes of the wild-type enzyme and the K151A and D180A mutants with L-2-chloropropionate were constructed by docking simulation. Subsequently, molecular dynamics were studied and ab initio fragment orbital calculations on the complexes were performed.…”

Theoretical prediction and experimental verification on enantioselectivity of haloacid dehalogenase l-DEX YL with chloropropionate

“…X-ray crystallographic analysis of l-DEX YL has provided information on the functions of active-site amino acid residues [7,8]. The contribution of each amino acid residue in l-DEX YL was examined through computer simulations with MD and FMO methods [14]. Recently, QM/MM simulations determined the transition state (TS) structure of the enzyme-substrate complex and elucidated its catalytic mechanism in the reaction of ester intermediate formation at the molecular level [15].…”

“…strain YL cell culture in a medium containing 2-chloropropionate (2-CPA) as the sole carbon source [5]. The reaction mechanism of l-DEX YL has been analyzed by site-directed mutagenesis [6], X-ray crystallography [7][8][9], mass spectrometry [10][11][12], 18 O-incorporation experiments [13], and computer simulations with molecular dynamics (MD), fragment molecular orbital (FMO) [14] and quantum-mechanical/molecularmechanical (QM/MM) methods [15]. Site-directed mutagenesis experiments have revealed that some amino acid residues are essential for the catalytic function of l-DEX YL [6].…”

Novel Approach for Identifying Key Residues in Enzymatic Reactions: Proton Abstraction in Ketosteroid Isomerase

“…33, 34 The FMO method has been applied to biomolecular systems aiming at better understanding of biological phenomena including enzymatic reactions [35][36][37][38] and at rational drug design etc.. In many FMO studies, binding modes of protein-small molecule (ligand or substrate), protein-protein, and protein-nucleic acid complexes have been investigated by analyzing mostly the following energy aspects: binding energies and energies of interactions between components of the complexes (for example, residue-residue interactions) at the molecular, atomic, and orbital levels.…”