Docking of cathenamine in THAS1-NADP+ structure

Cathenamine was docked into the THAS1-NADP⁺ crystal structure using Autodock 4.2 (ref. ⁶⁷). The ligand (cathenamine) was prepared with two torsions at the C16, the rest of the molecule being rigid and the receptor consisted of the desolvated high-resolution crystal structure. The search space was defined by a 40 × 40 × 40 Å box with a 0.375-Å grid spacing, centred between the nicotinamide ring and the side chain of Tyr56, and encompassed the entire active site cavity. Searches were performed using the Lamarckian Genetic Algorithm, consisting of 100 runs with a population size of 150 and 2,500,000 energy evaluations. A total of 27,000 generations were analysed and clustered with an RMS tolerance of 2 Å per cluster. This resulted in just two distinct clusters, which constituted 98% and 2% of the resultant poses, respectively. The latter cluster placed the indole moiety such that the nitrogen atom was closest to the cofactor. Thus, this cluster was eliminated since it was inconsistent with the results of the deuterium labelling experiments (Fig. 6). The poses contained within the major cluster were all deemed to be ‘productive' since they placed the indole moiety of cathenamine towards the entrance of the active site and the C20 and C21 3.3 Å above the nicotinamide C4 atom. The top ranked pose (with an estimated free energy of binding=−8.76 kcal mol⁻¹), as selected by the software, is used in the structures illustrated here (Fig. 4b).