Escherichia coli DH5α expressing recombinant dioxygenases from plasmids pKSJ90 (2NTDO M248I) and pKSJ92 (2NTDO L238V-M248I) were obtained from Rebecca E. Parales. Plasmids corresponding to pKMM32, pKMM33, and pKMM35 96 carrying the ntdAcAd genes for the I204A, I204T, and I204V variants of 2NTDO, respectively, were obtained from site-directed mutagenesis using pDTG800 carrying the ntdAcAd genes of wt 2NTDO as template. We performed site-directed mutagenesis using KLD mix (New England BioLabs) with forward primers including the exchanged codon and non-overlapping reverse primers that are listed in Table . Incubation and purification procedures followed the methods described for NBDO and 2NTDO. Activities, purities, and yields were comparable to those for 2NTDO.