Cloning and sequencing of cDNA encoding 4-aminobenzoate hydroxylase from an edible mushroom

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4-Aminobenzoate hydroxylase (EC 1.14.13.27) is a FAD-dependent monooxygenase which was found in an edible mushroom, Agaricus bisporus. We prepared three monoclonal antibodies recognizing the FAD-binding domain of the enzyme and showed that the antibodies can locate its FAD-binding domain. In order to elucidate the full primary structure of the enzyme, we tried to isolate a cDNA clone encoding the enzyme. The 613-bp DNA fragment encoding a part of the the enzyme was synthesized from poly(A)^+RNA by polymerase chain reaction and used as the probe in the following experiments. The size of the mRNA for the enzyme was shown to be approximately 1700 base by Northern blotting. DscDNA was prepared from poly(A)^+RNA,inserted into lambdagt10 arms, and then in vitro packaged. The cDNA library included 1*10^6 clones. Finally, four positive clones were isolated from the library by plaque hybridization. Of the clones, the clone with the longest insert cDNA was selected, the insert was subcloned into the Bam HI site of pUC19 plasmid and sequenced. The cDNA was shown to consist of 5'-untranslated region (14 bp), open reading frame (1380 bp), 3'-untranslated region (103 bp), and a poly A tail (20 bp) in a total nucleotide length of 1517 bp. The open reading frame coded for 460 amino acid protein with a molecular mass of 50974, and the deduced amino acid sequence included all of the amino acid sequences determined of 13 peptides which were isolated from the proteolytic products of the enzyme by high performance liquid chromatography. These findings demonstrate that the cDNA encodes the full length of the enzyme. At present, the development of an overexpression system of the cDNA is actively in progress.