Structural Evidence for the Dopamine-First Mechanism of Norcoclaurine Synthase

Abstract

Norcoclaurine synthase (NCS) is a Pictet-Spenglerase that catalyzes the first key step in plant benzylisoquinoline alkaloid metabolism, a compound family that includes bioactive natural products such as morphine. The enzyme has also shown great potential as a biocatalyst for the formation of chiral isoquinolines. Here we present new high-resolution X-ray crystallography data describing Thalictrum flavum NCS bound to a mechanism-inspired ligand. The structure supports two key features of the NCS "dopamine-first" mechanism: the binding of dopamine catechol to Lys-122 and the position of the carbonyl substrate binding site at the active site entrance. The catalytically vital residue Glu-110 occupies a previously unobserved ligand-bound conformation that may be catalytically significant. The potential roles of inhibitory binding and alternative amino acid conformations in the mechanism have also been revealed. This work significantly advances our understanding of the NCS mechanism and will aid future efforts to engineer the substrate scope and catalytic properties of this useful biocatalyst.

Figure 1

NCS-catalyzed reaction. (a) General reaction catalyzed by NCS. (b) NCS reactions with 4-hydroxyphenylacetaldehyde 2 (4-HPAA) and 4-methoxyphenylacetaldehyde (4-MOPAA) 3, and the intermediate mimic 6. (c) Outline of the proposed dopamine-first mechanism prior to these results.

Figure 2

Mimic bound in the active site of structure 5NON. (a) Overall structure, solvent-excluded surface, and active site entrance. The mimic is depicted as gray spheres. (b) The major conformation of the mimic is shown as gray sticks. Key active site side chains and loops are depicted. The distance between dopamine 3-OH and K122 (black dashed line) is 2.6 Å. The protein solvent-excluded surface has been clipped to show the active site cavity.

Figure 3

Interactions between catechol and K122. (a) Productive and (b) inhibitory mimic binding orientations, accounting for approximately 70 and 30% occupancy, respectively. The arrangement is productive when 3-OH is bound to K122. (c) Productive and (d) inhibitory dopamine binding orientation generated by computational docking. Numbers show the lengths in angstroms of the dotted lines.

Figure 4

Water in the active site. Water 201 interactions in (a) apo and (b) mimic-bound structures. (c) Quinone intermediate (light blue) docked into the 5NON active site (residues colored beige). Apo residues are colored gray to show proximity of E110 to C8a. Numbers are distances in angstroms. (d) Water channel mediated by hydrophilic active site residues. Gray sticks show the bound mimic; light blue sticks show (S)-hemiacetal docked into the active site, highlighting the position of the water leaving group. The protein solvent-excluded surface has been clipped to show the active site cavity.