The gene coding for the enzyme 2,3-dihydroxybenzoate-AMP ligase (2,3DHB-AMP ligase), responsible for activating 2,3-dihydroxybenzoic acid in the biosynthesis of the siderophore enterobactin, has been subcloned into the multicopy plasmid pKK223-3 and overproduced in a strain of Escherichia coli. The protein is an alpha 2 dimer with subunit molecular mass of 59 kDa. The enzyme catalyzes the exchange of [32P]pyrophosphate with ATP, dependent upon aromatic substrate with a turnover number of 340 min-1. The enzyme also releases pyrophosphate upon incubation with 2,3-dihydroxybenzoic acid and ATP; an initial burst corresponding to 0.7 nmol of pyrophosphate released per nanomole of enzyme is followed by a slower, continuous release with a turnover number of 0.41 min-1. The 1000-fold difference in rates observed between ATP-pyrophosphate exchange and continuous pyrophosphate release, as well as the close to stoichiometric amount of pyrophosphate released, suggests that intermediates are accumulating on the enzyme surface. Such intermediates have been observed and correspond to enzyme-bond (2,3-dihydroxybenzoyl)adenylate product.