Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-Cytochrome P450 reductases with P450 CYP73A5
Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-Cytochrome P450 reductases with P450 CYP73A5
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DOI：
10.1074/jbc.272.31.19176
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发表时间：
1997-08-01
影响因子：
4.8
通讯作者：
Pompon, D
中科院分区：
文献类型：
--
作者：
Urban, P;Mignotte, C;Pompon, D
通过对酿酒酵母cpr1基因突变株的代谢干扰，从拟南芥cdna文库中分离到两个编码NADPH-细胞色素P450还原酶的cDNAs，atr1编码692个氨基酸，而atr2编码712个残基的蛋白(atr2-1)或702个残基的蛋白(atr2-2)，这取决于起始密码子的选择。比较分析表明，atr1和atr2-1的氨基酸序列具有%的同源性，保守氨基酸密码子的第三个碱基没有保守性。这两个拟南芥还原酶由不同的基因编码，它们的差异可能是被子植物进化的早期事件，在ATR2-1的N端存在一个类似于植物叶绿体靶向信号的聚(Ser/Thr)拉伸，但在ATR1中不存在，在酵母中表达了cdna开放阅读框，重组多肽存在于酵母内质网膜中，具有高特异性的NADPH-细胞色素c还原酶活性。为了更深入地了解这两种还原酶各自的功能，我们克隆了拟南芥中编码肉桂酸4-羟基酶(CyP73A5)的基因，并在酵母中与ATR1或ATR2共表达对拟南芥ATR1/CYP73A5和ATR2-1/CYP73A5系统的生化鉴定表明，这两个远亲拟南芥还原酶类似地支持苯丙烷总途径的第一步氧化。
Two NADPH-cytochrome P450 reductase-encoding cDNAs were isolated from an Arabidopsis cDNA library by metabolic interference in a Saccharomyces cerevisiae mutant disrupted for its endogenous cpr1 gene, ATR1 encodes a protein of 692 amino acids, while ATR2 encodes either a 712-residue protein (ATR2-1), or a 702-residue protein (ATR2-2) depending on the choice of the initiation codon, Comparative analysis of ATR1 and ATR2-1 indicates 64% amino acid sequence identity and the absence of conservation in the third base of conserved amino acid codons. The two Arabidopsis reductases are encoded by distinct genes whose divergence is expected an early event in angiosperms evolution, A poly(Ser/Thr) stretch reminiscent of a plant chloroplastic targeting signal is present at the ATR2-1 N-terminal end but absent in ATR1, The cDNA open reading frames were expressed in yeast, The recombinant polypeptides were found present in the yeast endoplasmic reticulum membrane and exhibited a high specific NADPH-cytochrome c reductase activity, To gain more insight into the respective functions of the two reductases, the Arabidopsis cDNA encoding cinnamate 4-hydroxylase (CYP73A5) was cloned and co-expressed with ATR1 or ATR2 in yeast, Biochemical characterization of the Arabidopsis ATR1/CYP73A5 and ATR2-1/CYP73A5 systems demonstrates that the two distantly related Arabidopsis reductases similarly support the first oxidative step of the phenylpropanoid general pathway.