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 re combinant protein in Escherichia coli DH5alpha, led to the identificat ion 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 rati onal design of a series of random- and site-directed-mutagenesis exper iments of the Hdl IVa structural gene (hdl IVa). Subsequent apparent k inetic analyses of purified mutant enzymes identified His-20 and Arg-4 2 as the key residues in the activity of this halidohydrolase. It is a lso proposed that Asp-18 is implicated in the functioning of the enzym e, possibly by positioning the correct tautomer of His-20 for catalysi s in the enzyme-substrate complex and stabilizing the protonated form of His-20 in the transition-state complex. Comparison of conserved ami no acid sequences between the Hdl IVa and other halidohydrolases sugge sts that L-2-haloacid halidohydrolases contain conserved amino acid se quences that are not found in halidohydrolases active towards both D- and L-2-monochloropropionate.