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

4-氨基苯甲酸羟化酶（Aminobenzoate hydroxylase，EC 1.14.13.27）是一种存在于食用菌双孢蘑菇（Agaricus bisporus）中的FAD依赖性单加氧酶。我们制备了三个单克隆抗体识别的FAD结合域的酶，并表明，抗体可以定位其FAD结合域。为了阐明该酶的完整一级结构，我们试图分离编码该酶的cDNA克隆。通过聚合酶链反应，从poly（A）^+RNA中合成了编码部分酶的613 bp DNA片段，并将其用作以下实验中的探针。通过北方印迹显示该酶的mRNA大小约为1700个碱基。从poly（A）^+RNA中制备DscDNA，插入到引物10臂中，然后在体外包装。cDNA文库包括1*10^6个克隆。最后，通过噬斑杂交从文库中分离到4个阳性克隆。将插入片段插入pUC 19质粒的BamH Ⅰ位点，并进行序列测定。该cDNA由5 '非翻译区（14 bp）、开放阅读框（1380 bp）、3'非翻译区（103 bp）和polyA尾（20 bp）组成，全长1517 bp。该基因的开放阅读框编码460个氨基酸，分子量为50974，推导的氨基酸序列包含了高效液相色谱法分离的13个多肽的全部氨基酸序列。这些发现表明cDNA编码该酶的全长。目前，cDNA的过表达系统的开发正在积极进行中。

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.