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Article Dans Une Revue Biochemical Journal Année : 2008

Conformation, catalytic site, and enzymatic mechanism of the PR10 allergen related enzyme norcoclaurine synthase

Hanna Berkner
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Kristian Schweimer
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Irena Matecko
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Paul Rösch
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Résumé

The enzyme (S)-Norcoclaurine synthase (NCS; EC 4.2.1.78) found in the common meadow rue, Thalictrum flavum, and other plant species is involved in the biosynthesis of benzylisoquinoline alkaloids (BIAs). This group of plant secondary metabolites comprises pharmacologically active compounds like morphine and codeine. NCS catalyzes the condensation of 4-hydroxyphenylacetaldehyde (4-HPAA) and dopamine to (S)-norcoclaurine, the common precursor of all plant BIAs. While 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 class 10 of pathogenesis related proteins (PR10 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. While 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 size exclusion chromatography as well as NMR relaxation measurements, and the enzyme is predominantly monomer at the low micromolar concentrations used for activity assays.

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Dates et versions

hal-00478972 , version 1 (30-04-2010)

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Hanna Berkner, Kristian Schweimer, Irena Matecko, Paul Rösch. Conformation, catalytic site, and enzymatic mechanism of the PR10 allergen related enzyme norcoclaurine synthase. Biochemical Journal, 2008, 413 (2), pp.281-290. ⟨10.1042/BJ20080306⟩. ⟨hal-00478972⟩

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