Ounaroon Anan, Decker Gabriele, Schmidt Jürgen, Lottspeich Friedrich, Kutchan Toni M
Leibniz-Institut für Pflanzenbiochemie, Weinberg 3, D-06120 Halle/Saale, Germany.
Plant J. 2003 Dec;36(6):808-19. doi: 10.1046/j.1365-313x.2003.01928.x.
S-Adenosyl-L-methionine:(R,S)-reticuline 7-O-methyltransferase converts reticuline to laudanine in tetrahydrobenzylisoquinoline biosynthesis in the opium poppy Papaver somniferum. This enzyme activity has not yet been detected in plants. A proteomic analysis of P. somniferum latex identified a gel spot that contained a protein(s) whose partial amino acid sequences were homologous to those of plant O-methyltransferases. cDNA was amplified from P. somniferum RNA by reverse transcription PCR using primers based on these internal amino acid sequences. Recombinant protein was then expressed in Spodoptera frugiperda Sf9 cells in a baculovirus expression vector. Steady-state kinetic measurements with one heterologously expressed enzyme and mass spectrometric analysis of the enzymatic products suggested that this unusual enzyme is capable of carrying through sequential O-methylations on the isoquinoline and on the benzyl moiety of several substrates. The tetrahydrobenzylisoquinolines (R)-reticuline (4.2 sec(-1) mm(-1)), (S)-reticuline (4.5 sec(-1) mm(-1)), (R)-protosinomenine (1.7 sec(-1) mm(-1)), and (R,S)-isoorientaline (1.4 sec(-1) mm(-1)) as well as guaiacol (5.9 sec(-1) mm(-1)) and isovanillic acid (1.2 sec(-1) mm(-1)) are O-methylated by the enzyme with the ratio kcat/K m shown in parentheses. A P. somniferum cDNA encoding (R,S)-norcoclaurine 6-O-methyltransferase was similarly isolated and characterized. This enzyme was less permissive, methylating only (R,S)-norcoclaurine (7.4 sec(-1) mm(-1)), (R)-norprotosinomenine (4.1 sec(-1) mm(-1)), (S)-norprotosinomenine (4.0 sec(-1) mm(-1)) and (R,S)-isoorientaline (1.0 sec(-1) mm(-1)). A phylogenetic comparison of the amino acid sequences of these O-methyltransferases to those from 28 other plant species suggests that these enzymes group more closely to isoquinoline biosynthetic O-methyltransferases from Coptis japonica than to those from Thalictrum tuberosum that can O-methylate both alkaloid and phenylpropanoid substrates.
S-腺苷-L-甲硫氨酸：(R,S)-网状番荔枝碱7-O-甲基转移酶在罂粟（Papaver somniferum）的四氢苄基异喹啉生物合成过程中将网状番荔枝碱转化为劳丹碱。这种酶活性尚未在植物中被检测到。对罂粟乳汁进行的蛋白质组学分析鉴定出一个凝胶斑点，其中含有一种蛋白质，其部分氨基酸序列与植物O-甲基转移酶的氨基酸序列同源。基于这些内部氨基酸序列设计引物，通过逆转录PCR从罂粟RNA中扩增出cDNA。然后，重组蛋白在杆状病毒表达载体中于草地贪夜蛾（Spodoptera frugiperda）Sf9细胞中表达。对一种异源表达的酶进行稳态动力学测量以及对酶促产物进行质谱分析表明，这种不同寻常的酶能够对几种底物的异喹啉和苄基部分进行连续的O-甲基化。四氢苄基异喹啉（R）-网状番荔枝碱（4.2秒⁻¹毫摩尔⁻¹）、（S）-网状番荔枝碱（4.5秒⁻¹毫摩尔⁻¹）、（R）-原青藤碱（1.7秒⁻¹毫摩尔⁻¹）和（R,S）-异东方罂粟碱（1.4秒⁻¹毫摩尔⁻¹）以及愈创木酚（5.9秒⁻¹毫摩尔⁻¹）和异香草酸（1.2秒⁻¹毫摩尔⁻¹）被该酶进行O-甲基化，括号中显示的是催化常数与米氏常数的比值。一个编码（R,S）-去甲劳丹碱6-O-甲基转移酶的罂粟cDNA同样被分离并进行了表征。这种酶的选择性较低，仅能甲基化（R,S）-去甲劳丹碱（7.4秒⁻¹毫摩尔⁻¹）、（R）-去甲原青藤碱（4.1秒⁻¹毫摩尔⁻¹）、（S）-去甲原青藤碱（4.0秒⁻¹毫摩尔⁻¹）和（R,S）-异东方罂粟碱（1.0秒⁻¹毫摩尔⁻¹）。将这些O-甲基转移酶的氨基酸序列与其他28种植物的氨基酸序列进行系统发育比较表明，与来自日本黄连（Coptis japonica）的异喹啉生物合成O-甲基转移酶相比，这些酶与来自块茎唐松草（Thalictrum tuberosum）的能对生物碱和苯丙烷类底物都进行O-甲基化的O-甲基转移酶的亲缘关系更近。
