Alteration of cofactor specificity of the acrylyl-CoA reductase from Escherichia coli.

OBJECTIVES Alteration of the cofactor specificity of acrylyl-CoA reductase (AcuI) catalyzing the NAD(P)H-dependent reduction of acrylyl-CoA to propionyl-CoA is often desirable for designing of artificial metabolic pathways of various appointments. RESULTS Several variants of AcuIs from Escherichia coli K-12 with multiple amino acid substitutions to alter the cofactor preference were obtained by site directed mutagenesis and the modified enzymes as His6-tagged proteins were characterized. The simultaneous substitutions of arginine-180, arginine-198 and serine-178 residues by alanine in the enzyme pocket sequence as well as other amino acid changes decreased both NADPH- and NADH-dependent activities in comparison to the wild-type enzyme. The replacement of serine-156 by glutamic acid decreased NADPH-dependent activity at least 7000-fold but NADH-dependent activity only by threefold. The replacement of serine-156 by aspartic acid decreased NADPH-dependent activity 70-fold with fair preservation of activity and specificity to NADH. CONCLUSIONS These results demonstrated a relevance of Asp156 in the interaction of AcuI from E. coli K-12 with NADH as a coenzyme. These findings may provide reference information for shifting coenzyme specificity of acrylyl-CoA reductases.