Conformation, catalytic site, and enzymatic mechanism of the PR10 allergen-related enzyme norcoclaurine synthase
Conformation, catalytic site, and enzymatic mechanism of the PR10 allergen-related enzyme norcoclaurine synthase
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DOI：
10.1042/bj20080306
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发表时间：
2008-07-15
影响因子：
4.1
通讯作者：
Roesch, Paul
中科院分区：
文献类型：
--
作者：
Berkner, Hanna;Schweimer, Kristian;Roesch, Paul
在常见的草甸芸香、黄菊和其他植物物种中发现的酶NCS [（S）-去甲乌药碱合酶; EC 4.2.1.78]参与BIA（苄基异喹啉生物碱）的生物合成。这组植物次生代谢物包括药理活性化合物，如吗啡和可待因。NCS催化4-HPAA（4-羟基苯乙醛）和多巴胺缩合为（S）-去甲乌药碱，其为所有植物BIA的共同前体。虽然NCS的酶性质和反应的机理方面已被详细研究，但迄今为止还没有关于NCS的结构信息。该酶显示出与PR 10蛋白（致病相关蛋白的第10类）的成员（如主要的桦树花粉过敏原Bet v 1）的显着序列同源性。我们的CD和NMR光谱数据表明NCS和Bet v 1折叠的高度相似性，并允许我们使用Bet v 1作为模板来模拟NCS。通过NMR滴定实验，使用NCS序列的几乎完整的骨架分配来研究底物结合。虽然结合4-HPAA似乎诱导侧链重排的蛋白质的广泛的一部分，多巴胺的假定的独特的相互作用位点可以清楚地确定。NCS的寡聚化状态，据报道在酶的功能发挥了重要作用，被确定为浓度依赖性SEC（尺寸排阻色谱法）以及NMR弛豫测量，和酶被发现主要是一个单体在低微摩尔浓度用于活性测定。
The enzyme NCS [(S)-norcoclaurine synthase; EC 4.2.1.78] found in the common meadow rue, Thalictrum flavum, and other plant species, is involved in the biosynthesis of BIAs (benzylisoquinoline alkaloids). This group of plant secondary metabolites comprises pharmacologically-active compounds such as morphine and codeine. NCS catalyses the condensation of 4-HPAA (4-hydroxyphenylacetaldehyde) and dopamine to (S)-norcoclaurine, the common precursor of all plant BIAs. Although enzymatic properties of NCS and mechanistic aspects of the reaction have been studied in detail, no structural information on NCS was available so far. The enzyme shows significant sequence homology to members of the PR10 proteins (class 10 of pathogenesis-related proteins) such as the major birch pollen allergen Bet v 1. Our CD and NMR spectroscopic data indicated high similarity of the NCS and the Bet v 1 fold and allowed us to model NCS using Bet v 1 as a template. Virtually complete backbone assignment of the NCS sequence was used to study substrate binding by NMR titration experiments. Although binding of 4-HPAA seems to induce side-chain rearrangements in an extensive part of the protein, the putative distinct interaction site for dopamine could be clearly identified. The oligomerization state of NCS that reportedly plays an important role in enzyme functionality was determined to be concentration-dependent by SEC (size-exclusion chromatography) as well as NMR relaxation measurements, and the enzyme was found to be predominantly a monomer at the low micromolar concentrations used for activity assays.