Spectroscopic and computational studies of NTBC bound to the non-heme iron enzyme (4-hydroxyphenyl)pyruvate dioxygenase: Active site contributions to drug inhibition

Abstract

(4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) is an α-keto-acid-dependent dioxygenase which catalyzes the conversion of (4-hydroxyphenyl)pyruvate (HPP) to homogentisate as part of tyrosine catabolism. While several di- and tri-ketone alkaloids are known as inhibitors of HPPD and used commercially as herbicides, one such inhibitor, [2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC), has also been used therapeutically to treat type I tyrosinemia and alkaptonuria in humans. To gain further insight into the mechanism of inhibition by NTBC, a combination of CD/MCD spectroscopy and DFT calculations of HPPD/Fe II/NTBC has been performed to evaluate the contribution of the Fe II–NTBC bonding interaction to the high affinity of this drug for the enzyme. The results indicate that the bonding of NTBC to Fe II is very similar to that for HPP, both involving similar π-backbonding interactions between NTBC/HPP and Fe II. Combined with the result that the calculated binding energy of NTBC is, in fact, ∼3 kcal/mol less than that for HPP, the bidentate coordination of NTBC to Fe II is not solely responsible for its extremely high affinity for the enzyme. Thus, the π-stacking interactions between the aromatic rings of NTBC and two phenyalanine residues, as observed in the crystallography of the HPPD/Fe II/NTBC complex, appear to be responsible for the observed high affinity of drug binding.