Identification of functional residues essential for dehalogenation by the non-stereospecific α-haloalkanoic acid dehalogenase from Rhizobium sp. RC1.
The non-stereospecific α-haloalkanoic acid dehalogenase DehE from Rhizobium sp. RC1 catalyzes the removal of the halide from α-haloalkanoic acid D,L-stereoisomers and, by doing so, converts them into hydroxyalkanoic acid L,D-stereoisomers, respectively. DehE has been extensively studied to determine its potential to act as a bioremediation agent, but its structure/function relationship has not been characterized. For this study, we explored the functional relevance of several putative active-site amino acids by site-specific mutagenesis. Ten active-site residues were mutated individually, and the dehalogenase activity of each of the 10 resulting mutants in soluble cell lysates against D- and L-2-chloropropionic acid was assessed. Interestingly, the mutants W34→A,F37→A, and S188→A had diminished activity, suggesting that these residues are functionally relevant. Notably, the D189→N mutant had no activity, which strongly implies that it is a catalytically important residue. Given our data, we propose a dehalogenation mechanism for DehE, which is the same as that suggested for other non-stereospecific α-haloalkanoic acid dehalogenases. To the best of our knowledge, this is the first report detailing a functional aspect for DehE, and our results could help pave the way for the bioengineering of haloalkanoic acid dehalogenases with improved catalytic properties.
Department of Biotechnology, Kulliyyah of Science, IIUM, Bandar Indera Mahkota, Kuantan, Pahang, Malaysia.
Azzmer Azzar Abdul
AA
Hamid
Tengku Haziyamin Tengku Abdul
TH
Hamid
Roswanira Abdul
RA
Wahab
Fahrul
F
Huyop
D000595:Amino Acid Sequence; D055162:Biocatalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D054879:Halogenation; D006843:Hydrocarbons, Chlorinated / Q000378:metabolism*; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011422:Propionates / Q000378:metabolism*; D012231:Rhizobium / Q000201:enzymology* / Q000235:genetics*; D013237:Stereoisomerism; D013379:Substrate Specificity
D006843:Hydrocarbons, Chlorinated; D011422:Propionates; C021974:2-chloropropionic acid; D006867:Hydrolases; C034507:2-haloacid dehalogenase