Protein engineering of the 2-haloacid halidohydrolase IVa from Pseudomonas cepacia MBA4.
The chemical modification of L-2-haloacid halidohydrolase IVa (Hdl IVa), originally identified in Pseudomonas cepacia MBA4, produced as a recombinant protein in Escherichia coli DH5 alpha, led to the identification of histidine and arginine as amino acid residues likely to play a part in the catalytic mechanism of the enzyme. These results, together with DNA sequence and analyses [Murdiyatmo, Asmara, Baines, Bull and Hardman (1992) Biochem. J. 284, 87-93] provided the basis for the rational design of a series of random- and site-directed-mutagenesis experiments of the Hdl IVa structural gene (hdl IVa). Subsequent apparent kinetic analyses of purified mutant enzymes identified His-20 and Arg-42 as the key residues in the activity of this halidohydrolase. It is also proposed that Asp-18 is implicated in the functioning of the enzyme, possibly by positioning the correct tautomer of His-20 for catalysis in the enzyme-substrate complex and stabilizing the protonated form of His-20 in the transition-state complex. Comparison of conserved amino acid sequences between the Hdl IVa and other halidohydrolases suggests that L-2-haloacid halidohydrolases contain conserved amino acid sequences that are not found in halidohydrolases active towards both D- and L-2-monochloropropionate.
Asmara
W
W
International Institute of Biotechnology, University of Kent, Canterbury, U.K.
Murdiyatmo
U
U
Baines
A J
AJ
Bull
A T
AT
Hardman
D J
DJ
D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D016956:Burkholderia cepacia / Q000201:enzymology*; D003001:Cloning, Molecular; D004926:Escherichia coli; D006639:Histidine / Q000737:chemistry; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D015202:Protein Engineering
D009838:Oligodeoxyribonucleotides; D006639:Histidine; D001120:Arginine; D006867:Hydrolases; C034507:2-haloacid dehalogenase