Substrate binding in human indoleamine 2,3-dioxygenase 1: A spectroscopic analysis

•Residues Arg231 and Thr379 act in concert to ensure high affinity L-Trp binding in the hIDO active site.•Ser167 cannot be replaced by histidine to enhance hIDO substrate specificity.•4-phenyl-imidazole coordinates transiently to pentacoordinate ferrous heme iron, thus inhibiting the catalytic reaction.•Trp-like substrate analogs in the active site give rise to self-inhibition by blocking ligand access to the heme iron.

Human indoleamine 2,3-dioxygenase (hIDO1) is a heme enzyme that catalyzes the oxidative cleavage of the L-tryptophan indole ring. As increased levels of hIDO1 expression in tumor cells correlate with a poor prognosis for surviving several cancer types, hIDO1 has become an appealing drug target for cancer therapy. However, detailed structural knowledge of the catalytically active complex is necessary to eb able to design de novo inhibitors selective for hIDO1. Here we have applied Fourier transform infrared (FTIR) and nanosecond time-resolved optical spectroscopy to hIDO1 variants with modified heme pocket structures to identify important amino acid residues that stabilize the substrate in the active site. A cluster of small side chain residues at positions 260-265 ensures structural flexibility of the binding site. Thr379 and Arg231 are key residues acting in concert to bind the substrate. Thr379 is the final residue of a disordered loop; the neighboring Gly380, however, is still visible in the X-ray structure of the substrate-free protein, 20 Å away from the heme iron. Therefore, large-scale conformational changes are necessary to bring Thr379 close to the substrate. The use of substrate analogs further reveals that an indole-like side chain with two aromatic rings and L-stereoisomery at the Cα are required for high affinity binding.

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