Identification and characterization of CYP79D16 and CYP71AN24 catalyzing the first and second steps in l-phenylalanine-derived cyanogenic glycoside biosynthesis in the Japanese apricot, Prunus mume Sieb. et Zucc.
Identification and characterization of CYP79D16 and CYP71AN24 catalyzing the first and second steps in l-phenylalanine-derived cyanogenic glycoside biosynthesis in the Japanese apricot, Prunus mume Sieb. et Zucc.
复制标题
CYP79D16 和 CYP71AN24 的鉴定和表征，催化日本杏 Prunus mume Sieb 中 L-苯丙氨酸衍生的氰苷生物合成的第一步和第二步。
作者：
Takuya Yamaguchi;Kazunori Yamamoto;Y. Asano
日本杏、梅花Sieb.等，属于蔷薇科，产生作为防御剂的生氰苷普鲁纳辛和苦杏仁苷，这可能是从L苯丙氨酸。在本研究中，我们鉴定和表征了细胞色素P450催化L-苯丙氨酸转化为苯乙醛缩醛的反应。利用已公开的梅花核糖核酸测序数据，从‘南科’梅花中克隆了编码细胞色素P79D16、细胞色素P79A68、细胞色素P71AN24、细胞色素P71AP13、细胞色素P71AU50和细胞色素P736A117的全长基因，并进行了5‘和3’RACE。CYP79D16在幼苗中表达，而CYP71AN24在幼苗和叶片中表达。用液体和气相色谱-质谱法对这些细胞色素P450在酿酒酵母中表达的酶活性进行了鉴定。催化L-苯丙氨酸转化为苯乙醛的不是细胞色素P79A68，而是细胞色素P79D16。细胞色素P79D16对其他氨基酸无活性。催化苯乙醛肟合成扁桃腈的酶系为CYP71AN24，而不是CYP71AP13、CYP71AU50和CYP736A117。CYP71AN24还显示了各种芳香醛和腈的转化率较低。−-1的Km值为3.9m in，转化率为46.3m in。CYP71AN24的Km值和转换率可能导致苯乙醛肟的高效代谢，避免有毒中间体释放到胞浆中。这些结果表明，梅花中生氰糖苷的生物合成与CYP79D16在亲本反应和幼苗中的顺序反应中的催化作用是一致的。
Japanese apricot, Prunus mume Sieb. et Zucc., belonging to the Rosaceae family, produces as defensive agents the cyanogenic glycosides prunasin and amygdalin, which are presumably derived from l-phenylalanine. In this study, we identified and characterized cytochrome P450s catalyzing the conversion of l-phenylalanine into mandelonitrile via phenylacetaldoxime. Full-length cDNAs encoding CYP79D16, CYP79A68, CYP71AN24, CYP71AP13, CYP71AU50, and CYP736A117 were cloned from P. mume ‘Nanko’ using publicly available P. mume RNA-sequencing data, followed by 5′- and 3′-RACE. CYP79D16 was expressed in seedlings, whereas CYP71AN24 was expressed in seedlings and leaves. Enzyme activity of these cytochrome P450s expressed in Saccharomyces cerevisiae was evaluated by liquid and gas chromatography–mass spectrometry. CYP79D16, but not CYP79A68, catalyzed the conversion of l-phenylalanine into phenylacetaldoxime. CYP79D16 showed no activity toward other amino acids. CYP71AN24, but not CYP71AP13, CYP71AU50, and CYP736A117, catalyzed the conversion of phenylacetaldoxime into mandelonitrile. CYP71AN24 also showed lower conversions of various aromatic aldoximes and nitriles. The Km value and turnover rate of CYP71AN24 for phenylacetaldoxime were 3.9 µM and 46.3 min−1, respectively. The Km value and turnover of CYP71AN24 may cause the efficient metabolism of phenylacetaldoxime, avoiding the release of the toxic intermediate to the cytosol. These results suggest that cyanogenic glycoside biosynthesis in P. mume is regulated in concert with catalysis by CYP79D16 in the parental and sequential reaction of CYP71AN24 in the seedling.
影响因子：
2
作者：
Fukuda, T;Ito, H;Yoshida, T
通讯作者：
Yoshida, T