(4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) is a non-heme Fe(II)-dependent enzyme that catalyzes the second step in the tyrosine catabolism; in this step (4-hydroxyphenyl)pyruvate (HPP) is converted to 2,5-dihydroxyphenylacetate (homogentisate, HG). In humans, inhibitors of HPPD are used to alleviate symptoms of metabolic diseases such as type I tyrosinemia and alkaptonuria that arise from defects in the pathway. In plants, similar inhibitors are used successfully as herbicides as, in the plant, inhibition at this step prevents synthesis of redox cofactors vital to photosynthesis.

The first X-ray structure of HPPD was of the enzyme from Pseudomonas fluorescens. In this structure, the enzyme's C-terminal α-helix packs into the active site, blocking access to the metal ion. However, a number of recent structures have this α-helix displaced by the presence of inhibitory ligands. In the structure from Streptomyces avermitilis, the inhibitor molecule NTBC pivots the enzyme's C-terminal α-helix by 40° out of the active site into the solvent. In this position, Phe364 extends from the C-terminal helix to π-stack against the aromatic ring of the inhibitor. We have surmised that this interaction is important for the binding of inhibitors as well as the substrate HPP. It is apparent that the movement of the C-terminal α-helix at a minimum is required to gate ligand association. Moreover, a flexible loop tailing the C-terminal helix may be important in governing the movement of the helix itself. We present data from steady state and pre-steady state experiments that assess the kinetics and energetics of substrate binding using the Streptomyces avermitilis HPPD mutant in which this C-terminal loop has been deleted. The C-terminal deletion mutant binds substrate at least 6-orders of magnitude more slowly than does the wild type enzyme.