Conformation, catalytic site, and enzymatic mechanism of the PR10 allergen-related enzyme norcoclaurine synthase.
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.
Berkner
Hanna
H
Research Center for Bio-Macromolecules, Universität Bayreuth Universitätsstrasse 30, 95447 Bayreuth, Germany.
Schweimer
Kristian
K
Matecko
Irena
I
Rösch
Paul
P
D000485:Allergens / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D052179:Antigens, Plant; D001665:Binding Sites; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D002942:Circular Dichroism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary
D000485:Allergens; D000596:Amino Acids; D052179:Antigens, Plant; D010940:Plant Proteins; C061312:Bet v 1 allergen, Betula; D019731:Carbon-Nitrogen Ligases; C450500:norcoclaurine synthase