Berkner Hanna, Schweimer Kristian, Matecko Irena, Rösch Paul
Research Center for Bio-Macromolecules, Universität Bayreuth Universitätsstrasse 30, 95447 Bayreuth, Germany.
Biochem J. 2008 Jul 15;413(2):281-90. doi: 10.1042/BJ20080306.
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.
在普通唐松草（Thalictrum flavum）及其他植物物种中发现的NCS酶[（S）-去甲乌药碱合酶；EC 4.2.1.78]参与苄基异喹啉生物碱（BIA）的生物合成。这类植物次生代谢产物包含吗啡和可待因等具有药理活性的化合物。NCS催化4-羟基苯乙醛（4-HPAA）和多巴胺缩合生成（S）-去甲乌药碱，后者是所有植物BIA的共同前体。尽管已对NCS的酶学性质及反应的机制方面进行了详细研究，但目前尚无关于NCS的结构信息。该酶与病程相关蛋白10类（PR10蛋白）成员，如主要的桦树花粉过敏原Bet v 1，具有显著的序列同源性。我们的圆二色光谱和核磁共振光谱数据表明NCS与Bet v 1的折叠结构高度相似，这使我们能够以Bet v 1为模板对NCS进行建模。利用NCS序列几乎完整的主链归属，通过核磁共振滴定实验研究底物结合情况。尽管4-HPAA的结合似乎会在蛋白质的大部分区域诱导侧链重排，但可以清楚地识别出多巴胺假定的独特相互作用位点。据报道，NCS的寡聚化状态在酶功能中起重要作用，通过尺寸排阻色谱（SEC）以及核磁共振弛豫测量确定其为浓度依赖性，并且发现在用于活性测定的低微摩尔浓度下，该酶主要为单体。
