Cloning and analysis of the Genes Responsible for Biosynthesis of Norspermidine in Vibrio

1.确定酶的N-末端氨基酸序列并制备针对酶的抗体。纯化了溶藻弧菌中三种参与多胺合成的新酶，并测定了它们的N-末端氨基酸序列。成功地测定了每种酶的10 - 20个氨基酸残基。针对这些酶产生的抗体对相应的酶具有特异性.羧基去甲亚精胺脱羧酶基因的克隆和表达。用免疫学方法从基因文库中筛选出16个阳性克隆，其中一个携带重组质粒pCDC 14的克隆酶活比溶藻弧菌粗提液酶活高3倍。对大肠杆菌细胞粗提物进行Western印迹分析。pCDC 14在大肠杆菌HB 101中表达，其相对分子质量为43500，与溶藻弧菌CANS DC的相对分子质量相当。插入该片段 关于我们 （4kbp）反向插入pUC 18，酶活性没有变化，表明包括启动子区的整个基因被克隆。对缺失质粒进行亚克隆，将该基因定位于一个长度为2kbp的片段（pCDC 14 -1）.编码CANS DC的基因的核苷酸序列。从pCDC 14 -1制备巢式缺失质粒以确定该基因的核苷酸序列。该基因上游区域的特征在于推定的启动子共有区（-10，-35），核糖体结合位点和ATG起始密码子。从起始于ATG的核苷酸序列推导的氨基酸序列完全对应于已经确定的N-末端氨基酸序列。此外，还鉴定了终止密码子TAA，随后是类似于p -非依赖性转录终止的结构的序列。我们发现，与溶藻弧菌一样，醋酸钙不动杆菌也具有L-2，4-二氨基丁酸脱羧酶（DABA DC），它催化丙烷二氨基的产生，丙烷二氨基在该菌中大量存在。结果表明，该酶在亚基结构、N端氨基酸序列和免疫反应性等方面与溶藻弧菌的DABA DC不同。少

1. Determination of N-terminal amino acid sequences of the enzymes and preparation of the antibodies against the enzymes. Three novel enzymes involved in biosynthesis of a polyamine, norspermidine, in Vibrio alginolyticus were purified to determine their N-terminal amino acid sequences. Ten to 20 amino acid residues of each enzyme were successfully determined. The antibodies raised against these enzymes were specific for corresponding enzymes.2. Cloning and expression of the gene encoding carboxynorspermidine decarboxylase(CANS DC). Sixteen positive clones were selected from the gene library by the immunological method, and one clone haboring a recombinant plasmid, designated pCDC14, showed the enzyme activity 3-fold higher than that in the V. alginolyticus crude cell extract. Western blot analysis of a crude cell extract of E. coli HB101 haboring pCDC14 demonstrated a protein band with an M_r of 43500, which was comparable with CANS DC from V. alginolyticus. Insertion of this fragment … More (4 kbp)into pUC18 in a reverse orientation resulted in no change in enzyme activity, suggesting that the whole gene including the promoter region is cloned. Subcloning of the deleted plasmids localized the gene in a fragment of 2 kbp in length(pCDC14-1).3. Nucleotide sequence of the gene encoding CANS DC. The nested deletion plasmids were prepared from pCDC14-1 to determine the nucleotide sequence of this gene. The region upstream from the gene was characterized by a putative promoter consensus region(-10, -35), a ribosome-binding site and ATG start codon. The amino acid sequence deduced from the nucleotide sequence beginning at ATG completely corresponded to the N-terminal amino acid sequence already determined. Moreover, the termination codon TAA followed by a sequence for a structure resembling a p -independent transcription termination ws also identified.4. We have found that, like V. alginolyticus, Acinetobacter calcoaceticus possesses L-2, 4diaminobuty-rate decarboxylase(DABA DC)catalyzing the production of diaminopropane, which is present in this bactrium in a large amount. It was found that the purified enzyme was different in subunit structure, N-terminal amino acid sequence and immunoreactivity from the DABA DC of V. alginolyticus. Less