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

4-氨基苯甲酸酯羟化酶（EC 1.14.13.27）是在双孢蘑菇中发现的一种fad依赖性单加氧酶。我们制备了三种识别该酶的fad结合域的单克隆抗体，并证明抗体可以定位其fad结合域。为了阐明该酶的完整一级结构，我们试图分离一个编码该酶的cDNA克隆。通过聚合酶链反应，以poly(a)^+RNA为原料合成了编码部分酶的613 bp DNA片段，并作为后续实验的探针。该酶mRNA的大小通过Northern blotting显示约为1700个碱基。用poly(A)^+RNA制备DscDNA，插入到lambdagt10臂中，然后进行体外包装。cDNA文库包含1*10^6个克隆。最后通过菌斑杂交从文库中分离出4个阳性克隆。选择插入cDNA最长的克隆，将其亚克隆到pUC19质粒的Bam HI位点并测序。cDNA全长1517 bp，由5′-非翻译区（14 bp）、开放阅读框（1380 bp）、3′-非翻译区（103 bp）和一个聚a尾（20 bp）组成。该开放阅读框编码了460个分子量为50974的氨基酸蛋白，推导出的氨基酸序列包含了从该酶蛋白水解产物中分离得到的13个肽的全部氨基酸序列。这些发现表明，cDNA编码的酶的全部长度。目前，cDNA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.