Burga Laura, Wellmann Frank, Lukacin Richard, Witte Simone, Schwab Wilfried, Schröder Joachim, Matern Ulrich
Institut für Pharmazeutische Biologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, D-35037 Marburg, Germany.
Arch Biochem Biophys. 2005 Aug 1;440(1):54-64. doi: 10.1016/j.abb.2005.05.026.
A cDNA was cloned from Ruta graveolens cells encoding a novel O-methyltransferase (OMT) with high similarity to orcinol or chavicol/eugenol OMTs, but containing a serine-rich N-terminus and a 13 amino acid insertion between motifs IV and V. Expression in Escherichia coli revealed S-adenosyl-l-methionine-dependent OMT activity with methoxylated phenols only with an apparent Km of 20.4 for the prime substrate 3,5-dimethoxyphenol. The enzyme forms a homodimer of 84 kDa, and the activity was insignificantly affected by 2.0 mM Ca2+ or Mg2+, whereas Fe2+, Co2+, Zn2+, Cu2+ or Hg2+ were inhibitory (78-100%). Dithiothreitol (DTT) suppressed the OMT activity. This effect was examined further, and, in the presence of Zn2+ as a potential thiol methyltransferase (TMT) cofactor, the recombinant OMT methylated DTT to DTT-monomethylthioether. Sets of kinetic OMT experiments with 3,5-dimethoxyphenol at various Zn2+/DTT concentrations revealed the competitive binding of DTT with an apparent Ki of 52.0 microM. Thus, the OMT exhibited TMT activity with almost equivalent affinity to the thiol pseudosubstrate which is structurally unrelated to methoxyphenols.
从芸香细胞中克隆出一个cDNA，其编码一种新型O-甲基转移酶（OMT），该酶与苔黑酚或查维酚/丁香酚OMT具有高度相似性，但N端富含丝氨酸，且在基序IV和V之间有13个氨基酸的插入序列。在大肠杆菌中的表达显示，该酶具有依赖S-腺苷-L-甲硫氨酸的OMT活性，仅对甲氧基化酚有活性，对主要底物3,5-二甲氧基苯酚的表观Km为20.4。该酶形成一个84 kDa的同型二聚体，2.0 mM的Ca2+或Mg2+对其活性影响不显著，而Fe2+、Co2+、Zn2+、Cu2+或Hg2+具有抑制作用（78 - 100%）。二硫苏糖醇（DTT）抑制OMT活性。对此效应进行了进一步研究，在存在作为潜在硫醇甲基转移酶（TMT）辅因子的Zn2+时，重组OMT将DTT甲基化为DTT-单甲硫醚。在不同Zn2+/DTT浓度下用3,5-二甲氧基苯酚进行的一系列动力学OMT实验表明，DTT具有竞争性结合，表观Ki为52.0 μM。因此，该OMT表现出TMT活性，对与甲氧基苯酚结构无关的硫醇假底物具有几乎相同的亲和力。
