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

Hamid AA, Hamid TH, Wahab RA, Omar MS, Huyop F - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Faculty of Science, IIUM, Bandar Indera Mahkota, 25200, Kuantan, Pahang, Malaysia.

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.

No MeSH data available.


The total energies of S188V-3CP and DehE-3CP during 10,000-ps simulations.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4376737&req=5

pone.0121687.g009: The total energies of S188V-3CP and DehE-3CP during 10,000-ps simulations.

Mentions: At the beginning of the trajectory (t = 0 ps) until ~5,500 ps, RMSD of the S188V-3CP complex was lower (1.0–2.0 Å) than for DehE-3CP (1.25–2.5 Å) (Fig. 8). Deviation values for both complexes rapidly increased during the initial 5,500 ps, indicating dynamic structural changes during the equilibration process. Between 5,500 and 7,500 ps, the RMSDs of S188V-3CP and DehE-3CP were stable at ~2.0 Å. After 7,500 ps, however, the deviation value for S188V-3CP began to decrease, whereas the DehE-3CP RMSD remained at ~2.25 Å. The difference in deviation values between the complexes after 7,500 ps could be attributed to structural changes associated with the S188V mutation. Concerning energy plots, S188V-3CP possessed a low total energy of approximately—8.4* 105 kJ/mol (Fig. 9). As such, this mutation significantly reduced the average total energy of S188–3CP compared with other DehE complexes. This likely resulted from the stabilisation of bonds between the carboxylate group of 3CP and the backbone of the valine residue within the DehE active site.


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

Hamid AA, Hamid TH, Wahab RA, Omar MS, Huyop F - PLoS ONE (2015)

The total energies of S188V-3CP and DehE-3CP during 10,000-ps simulations.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4376737&req=5

pone.0121687.g009: The total energies of S188V-3CP and DehE-3CP during 10,000-ps simulations.
Mentions: At the beginning of the trajectory (t = 0 ps) until ~5,500 ps, RMSD of the S188V-3CP complex was lower (1.0–2.0 Å) than for DehE-3CP (1.25–2.5 Å) (Fig. 8). Deviation values for both complexes rapidly increased during the initial 5,500 ps, indicating dynamic structural changes during the equilibration process. Between 5,500 and 7,500 ps, the RMSDs of S188V-3CP and DehE-3CP were stable at ~2.0 Å. After 7,500 ps, however, the deviation value for S188V-3CP began to decrease, whereas the DehE-3CP RMSD remained at ~2.25 Å. The difference in deviation values between the complexes after 7,500 ps could be attributed to structural changes associated with the S188V mutation. Concerning energy plots, S188V-3CP possessed a low total energy of approximately—8.4* 105 kJ/mol (Fig. 9). As such, this mutation significantly reduced the average total energy of S188–3CP compared with other DehE complexes. This likely resulted from the stabilisation of bonds between the carboxylate group of 3CP and the backbone of the valine residue within the DehE active site.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Faculty of Science, IIUM, Bandar Indera Mahkota, 25200, Kuantan, Pahang, Malaysia.

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.

No MeSH data available.