Octopine and nopaline oxidases from Ti plasmids of Agrobacterium tumefaciens: molecular analysis, relationship, and functional characterization
Octopine and nopaline oxidases from Ti plasmids of Agrobacterium tumefaciens: molecular analysis, relationship, and functional characterization
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根癌农杆菌 Ti 质粒中的章鱼碱和胭脂碱氧化酶：分子分析、关系和功能表征
DOI：
10.1128/jb.176.15.4511-4517.1994
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发表时间：
1994
影响因子：
3.2
通讯作者：
J. Schröder
中科院分区：
文献类型：
--
作者：
H. Zanker;G. Lurz;U. Langridge;P. Langridge;D. Kreusch;J. Schröder
PTiAch5(章鱼碱)和pTiC58(萝卜碱)钛质粒的OCC和NOC区负责章鱼碱和萝卜碱在农杆菌中的分解代谢。酶促反应的第一步是膜结合的酪氨酸氧化酶将L精氨酸和丙酮酸或2-酮戊二酸分别氧化裂解成L精氨酸和丙酮酸或2-酮戊二酸，需要两个多肽(亚基B和A)发挥作用。DNA测序表明，pTiAch5和pTiC58的亚基是相关的，但没有一个蛋白与转化植物细胞中表达的生物合成酶有显著的相似性。这四个蛋白质与其他蛋白质总体上没有广泛的相似性，但35个N末端氨基酸包含许多酶中的基序，这些基序利用黄素腺嘌呤二核苷酸、黄素单核苷酸或NAD(P)+作为辅因子。然而，活性与添加的辅因子完全无关，电子受体的性质尚不清楚。膜的增溶导致酶活性的完全丧失。鸦片碱氧化酶可接受鸦片碱和章鱼碱(Vmax比为5：1)，Km值相近(1.1 mM)。章鱼碱氧化酶对章鱼碱(Km=1 mM)有很高的活性，而对诺帕林几乎检测不到活性。OCC区和NOC区的亚基是可以互换的。组合ooxB-noxA和noxB-ooxA都产生了活性酶，其氧化章鱼碱和萝卜碱的速率相似，这表明这两个亚基对底物专一性有贡献。这些实验还表明，功能性酶的形成需要同一质粒上的亚基基因非常接近，即使基因顺序颠倒(A-B而不是B-A)也会导致活性降低。
The occ and noc regions of pTiAch5 (octopine) and pTiC58 (nopaline) Ti plasmids are responsible for the catabolic utilization of octopine and nopaline in Agrobacterium spp. The first enzymatic step is the oxidative cleavage into L-arginine and pyruvate or 2-ketoglutarate, respectively, by membrane-bound opine oxidases requiring two polypeptides (subunits B and A) for function. The DNA sequences showed that the subunits of pTiAch5 and pTiC58 are related, but none of the proteins revealed significant similarities to the biosynthetic enzymes expressed in transformed plant cells. The four proteins had no extensive overall similarity to other proteins, but the 35 N-terminal amino acids contained motifs found in many enzymes utilizing flavin adenine dinucleotide, flavin mononucleotide, or NAD(P)+ as cofactors. However, the activities were completely independent of added cofactors, and the nature of the electron acceptor remained unclear. Membrane solubilization led to complete loss of enzyme activity. The nopaline oxidase accepted nopaline and octopine (Vmax ratio, 5:1) with similar Km values (1.1 mM). The octopine oxidase had high activity with octopine (Km = 1 mM) and barely detectable activity with nopaline. The subunits from the occ and the noc regions were exchangeable. The combinations ooxB-noxA and noxB-ooxA both produced active enzymes which oxidized octopine and nopaline at similar rates, suggesting that both subunits contributed to the substrate specificity. These experiments also showed that the formation of functional enzyme required close proximity of the subunit genes on the same plasmid and that even a reversal of the gene order (A-B instead of B-A) led to reduced activity.