Wu Tianyun, Yankovskaya Victoria, McIntire William S
Molecular Biology Division of the Department of Veterans Affairs Medical Center, San Francisco, the Northern California Institute for Research and Education, San Francisco, California 94121, USA.
J Biol Chem. 2003 Jun 6;278(23):20514-25. doi: 10.1074/jbc.M302149200. Epub 2003 Mar 26.
The aminoacyl sequences of three regions of pure bovine N1-acetylated polyamine oxidase (PAO) were obtained and used to search GenBankTM. This led to the cloning and sequencing of a complete coding cDNA for murine PAO (mPAO) and the 5'-truncated coding region of the bovine pao (bpao) gene. A search of GenBankTM indicated that mpao maps to murine chromosome 7 as seven exons. The translated amino acid sequences of mpao and bpao have a -Pro-Arg-Leu peroxisomal targeting signal at the extreme C termini. A beta-alpha-beta FAD-binding motif is present in the N-terminal portion of mPAO. This and several other regions of mPAO and bPAO are highly similar to corresponding sections of other flavoprotein amine oxidases, although the overall identity of aligned sequences indicates that PAO represents a new subfamily of flavoproteins. A fragment of mpao was used as a probe to establish the relative transcription levels of this gene in various mature murine tissues and murine embryonic and breast tissues at different developmental stages. An Escherichia coli expression system has been developed for manufacturing mPAO at a reasonable level. The mPAO so produced was purified to homogeneity and characterized. It was demonstrated definitively that PAO oxidizes N1-acetylspermine to spermidine and 3-acetamidopropanal and that it also oxidizes N1-acetylspermidine to putrescine and 3-acetamidopropanal. Thus, this is the classical polyamine oxidase (EC 1.5.3.11) that is defined as the enzyme that oxidizes these N1-acetylated polyamines on the exo-side of their N4-amino groups. This enzyme is distinguishable from the plant polyamine oxidase that oxidizes spermine on the endo-side of the N4-nitrogen. It differs also from mammalian spermine oxidase that oxidizes spermine (but not N1-acetylspermine or N1-acetylspermidine) at the exo-carbon of its N4-amino group. This report provides details of the biochemical, spectral, oxidation-reduction, and steady-state kinetic properties of pure mPAO.
获得了纯牛N1 - 乙酰化多胺氧化酶（PAO）三个区域的氨酰基序列，并用于搜索GenBankTM。这导致了小鼠PAO（mPAO）完整编码cDNA以及牛pao（bpao）基因5'端截短编码区的克隆和测序。对GenBankTM的搜索表明，mpao作为七个外显子定位于小鼠7号染色体。mpao和bpao的翻译氨基酸序列在极端C末端具有-Pro-Arg-Leu过氧化物酶体靶向信号。mPAO的N端部分存在β-α-β FAD结合基序。mPAO和bPAO的这一区域以及其他几个区域与其他黄素蛋白胺氧化酶的相应部分高度相似，尽管比对序列的总体一致性表明PAO代表黄素蛋白的一个新亚家族。一段mpao片段用作探针，以确定该基因在各种成熟小鼠组织以及不同发育阶段的小鼠胚胎和乳腺组织中的相对转录水平。已开发出一种大肠杆菌表达系统，用于以合理水平生产mPAO。如此产生的mPAO被纯化至同质并进行了表征。已明确证明PAO将N1 - 乙酰精胺氧化为亚精胺和3 - 乙酰氨基丙醛，并且它还将N1 - 乙酰亚精胺氧化为腐胺和3 - 乙酰氨基丙醛。因此，这就是经典的多胺氧化酶（EC 1.5.3.11），其被定义为在其N4 - 氨基的外侧氧化这些N1 - 乙酰化多胺的酶。这种酶与在N4 - 氮的内侧氧化精胺的植物多胺氧化酶不同。它也与在其N4 - 氨基的外侧碳上氧化精胺（但不氧化N1 - 乙酰精胺或N1 - 乙酰亚精胺）的哺乳动物精胺氧化酶不同。本报告提供了纯mPAO的生化、光谱、氧化还原和稳态动力学特性的详细信息。
