Reduction of carboxylic acids by Nocardia aldehyde oxidoreductase requires a phosphopantetheinylated enzyme.
Aldehyde oxidoreductase (carboxylic acid reductase (Car)) catalyzes the magnesium-, ATP-, and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes. Heterologous expression of the car gene in Escherichia coli afforded purified recombinant enzyme with a specific activity nearly 50-fold lower than that of purified native Nocardia sp. enzyme. The 5-fold increase in specific activity obtained by incubating purified recombinant Car with CoA and Nocardia cell-free extracts indicated that post-translational phosphopantetheinylation of Car is required for maximum enzyme activity. Nocardia phosphopantetheine transferase (PPTase) expressed in E. coli was isolated and characterized. When incubated with [(3)H]acetyl-CoA and Nocardia PPTase, the labeled acetylphosphopantetheine moiety was incorporated into recombinant Car. Coexpression of Nocardia Car and PPTase in E. coli gave a reductase with nearly 20-fold higher specific activity. Site-directed mutagenesis in which Ser(689) was replaced with Ala resulted in an inactive Car mutant. The results show that Car expressed in Escherichia coli is an apoenzyme that is converted to a holoenzyme by post-translational modification via phosphopantetheinylation. Doubly recombinant resting E. coli cells efficiently reduce vanillic acid to vanillin.
Venkitasubramanian
Padmesh
P
Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa 52242-5000, USA.
Daniels
Lacy
L
Rosazza
John P N
JP
D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000502:physiology*; D001426:Bacterial Proteins / Q000737:chemistry; D001547:Benzaldehydes / Q000737:chemistry; D001592:Benzyl Alcohols / Q000737:chemistry; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D008954:Models, Biological; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D025102:Myosin Type III / Q000737:chemistry; D009615:Nocardia / Q000201:enzymology*; D010088:Oxidoreductases / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry
D001426:Bacterial Proteins; D001547:Benzaldehydes; D001592:Benzyl Alcohols; D002264:Carboxylic Acids; D011994:Recombinant Proteins; C082662:phosphopantetheinyl transferase; C100058:vanillin; D010088:Oxidoreductases; D000445:Aldehyde Oxidoreductases; C019825:carboxylic acid reductase; D017855:Transferases (Other Substituted Phosphate Groups); D025102:Myosin Type III; C024078:vanillyl alcohol