Putative ACP Phosphodiesterase Gene (acpD) Encodes an Azoreductase

An FMN-dependent NADH-azoreductase ofEscherichia coli was purified and analyzed for identification of the gene responsible for azo reduction by microorganisms. The N-terminal sequence of the azoreductase conformed to that of the acpD gene product, acyl carrier protein phosphodiesterase. Overexpression of the acpD gene provided the E. coli with a large amount of the 23-kDa protein and more than 800 times higher azoreductase activity. The purified gene product exhibited activity corresponding to that of the native azoreductase. The reaction followed a ping-pong mechanism requiring 2 mol of NADH to reduce 1 mol of methyl red (4′-dimethylaminoazobenzene-2-carboxylic acid) into 2-aminobenzoic acid and N,N′-dimethyl-p-phenylenediamine. On the other hand, the gene product could not convert holo-acyl carrier protein into the apo form under either in vitro or in vivo conditions. These data indicate that the acpDgene product is not acyl carrier protein phosphodiesterase but an azoreductase.