An S188V mutation alters substrate specificity of non-stereospecific α-haloalkanoic acid dehalogenase E (DehE)

Abstract

The non-stereospecific α-haloalkanoic acid dehalogenase E (DehE) degrades many halogenated compounds but is ineffective against β-halogenated compounds such as 3-chloropropionic acid (3CP). Using molecular dynamics (MD) simulations and site-directed mutagenesis we show here that introducing the mutation S188V into DehE improves substrate specificity towards 3CP. MD simulations showed that residues W34, F37, and S188 of DehE were crucial for substrate binding. DehE showed strong binding ability for D-2-chloropropionic acid (D-2CP) and L-2-chloropropionic acid (L-2CP) but less affinity for 3CP. This reduced affinity was attributed to weak hydrogen bonding between 3CP and residue S188, as the carboxylate of 3CP forms rapidly interconverting hydrogen bonds with the backbone amide and side chain hydroxyl group of S188. By replacing S188 with a valine residue, we reduced the inter-molecular distance and stabilised bonding of the carboxylate of 3CP to hydrogens of the substrate-binding residues. Therefore, the S188V can act on 3CP, although its affinity is less strong than for D-2CP and L-2CP as assessed by Km. This successful alteration of DehE substrate specificity may promote the application of protein engineering strategies to other dehalogenases, thereby generating valuable tools for future bioremediation technologies.

Fig 1. HPLC profile of 3HP (t_R: 21.85) and 3CP (t_R: 23.92) standards (10 mM each).

Fig 2. Root mean square deviations (RMSDs) of the alpha-carbon positions during 10,000-ps simulations.

Fig 3. Molecular visualisation of substrate binding.

(A) d-2CP in the binding pocket of DehE. (B) Hydrogen bonding between d-2CP and W34, F37, and S188 in the DehE-d-2CP complex. (C) l-2CP in binding pocket of DehE. (D) Hydrogen bonding between l-2CP and W34, F37, and S188 in the DehE-l-2CP complex. (E) 3CP in the binding pocket of DehE. (F) Hydrogen bonding between 3CP and W34, F37, and S188 in the DehE-3CP complex.

Fig 4. Root mean square fluctuations (RMSFs) of the alpha-carbon positions of DehE complexes.

Fig 5. Total energy of DehE complexes during 10,000-ps simulations.

Fig 6. Atomic distance analysis of MD trajectories associated with each of the DehE-d-2CP, DehE-l-2CP, and DehE-3CP complexes.

(A) Time course of distances between HE1 of W34 and O of d-2CP (purple line), HE1 of W34 and O of l-2CP (green line), and HE1 of W34 and O of 3CP (red line). (B) Time course of distances between H of F37 and O of d-2CP (purple line), H of F37 and O of l-2CP (green line), and H of F37 and O of 3CP (red line). (C) Time course of distances between H of S188 and O^- of d-2CP (purple line), H of S188 and O^- of l-2CP (green line), and H of S188 and O^- of 3CP O^- (red line).

Fig 7. Interconversion of hydrogen bonds between the 3CP carboxylate and S188 in DehE-3CP complex.

Fig 8. Root mean square deviations (RMSDs) of the alpha-carbon positions for S188V-3CP and DehE-3CP during 10,000-ps simulations.

Fig 9. The total energies of S188V-3CP and DehE-3CP during 10,000-ps simulations.

Fig 10. Atomic distance analysis of the MD trajectory of DehE-3CP and S188V-3CP complexes.

(A) Time courses of the distance between HE1 of W34 and O of 3CP for DehE-3CP (red line) and HE1 of W34 and O of 3CP for S188V-3CP (blue line). (B) Time courses of the distance between H of F37 and O of 3CP for DehE-3CP (red line) and of H of F37 and O of 3CP for S188V-3CP (blue line). (C) Time courses of the distance between H of S188 and O^-of 3CP for DehE-3CP (red line) and of H of V188 and O^-of 3CP for S188V-3CP (blue line)

Fig 11. HPLC profile of 3CP degradation.

(a): degradation of 3CP producing 3HP (t_R 21.86). (b) The 3CP (t_R: 23.94) was not degraded by the enzyme in cell free extract from the wild type DehE.

Fig 12. SDS-PAGE analysis of cell free extracts and pure proteins.

a) Lane 1: Protein markers (Invitrogen); Lane 2: cell free extract of wild type DehE; Lane 3: S188V DehE mutant. b) Lane 4: pure enzyme extract of wild type DehE (32 kDa) Lane 5: pure enzyme extract of S188V DehE mutant (32 kDa); Lane 6: Protein markers (Invitrogen).

Fig 13. A possible reaction mechanism of hydrolysis catalysed by DehE mutant. Asp189 incorporated with Asn 114 both act as base at the DehE enzyme structure.

This initiate water activation. The nucleophilic attack of the carbon-halogen bond of the substrate takes place and resulted in formation of intermediate. Finally, the release of halide (chloride ion) from the compound and formation of hydroxylated product can be detected.