The Interactions in the Carboxyl Terminus of Human 4-Hydroxyphenylpyruvate Dioxygenase Are Critical to Mediate the Conformation of the Final Helix and the Tail to Shield the Active Site for Catalysis

Abstract

4-Hydroxylphenylpyruvate dioxygenase (4-HPPD) is an important enzyme for tyrosine catabolism, which catalyzes the conversion of 4-hydroxylphenylpyruvate (4-HPP) to homogentisate. In the present study, human 4-HPPD was cloned and expressed in E. coli. The kinetic parameters for 4-HPP conversion were: k cat = 2.2±0.1 s−1; and K m = 0.08±0.02 mM. Sequence alignments show that human 4-HPPD possesses an extended C-terminus compared to other 4-HPPD enzymes. Successive truncation of the disordered tail which follows the final α-helix resulted in no changes in the K m value for 4-HPP substrate but the k cat values were significantly reduced. The results suggest that this disordered C-terminal tail plays an important role in catalysis. For inspection the effect of terminal truncation on protein structure, mutant models were built. These models suggest that the different conformation of E254, R378 and Q375 in the final helix might be the cause of the activity loss. In the structure E254 interacts with R378, the end residue in the final helix; mutation of either one of these residues causes a ca. 95% reductions in k cat values. Q375 provides bifurcate interactions to fix the tail and the final helix in position. The model of the Q375N mutant shows that a solvent accessible channel opens to the putative substrate binding site, suggesting this is responsible for the complete loss of activity. These results highlight the critical role of Q375 in orientating the tail and ensuring the conformation of the terminal α-helix to maintain the integrity of the active site for catalysis.

Structurally diverse dehydroshikimate dehydratase variants participate in microbial quinate catabolism

“…A). This region has been observed to exhibit a significant level of mobility in other HPPD structures, and may serve a gating function for active site ligands (Brownlee et al ., ; Lin et al ., ).…”

The different catalytic roles of the metal-binding ligands in human 4-hydroxyphenylpyruvate dioxygenase

“…The variants of H183A, H266A, E349A, E349G, and E349Q were generated by using the QuikChange mutagenesis kit with Pfu DNA polymerase and the vector of pTrc-HPPD as template [41]. Two complementary primers including the desired mutations were used for PCR (Table S1).…”

“…The overexpression and purification of recombinant wild-type and variant HPPD enzymes were carried out as reported previously [41]. In brief, the supernatant of crude cell extract were loaded onto Q-Sepharose anion exchanger column (26 x 150 mm) equilibrated in 50 mM Tris-HCl buffer, pH 7.5, 1 mM EDTA and 1 mM DTT, and eluted with the same buffer.…”

“…The activity of HPPD was measured using both the Oxygraph and HPLC assays [41]. The oxygen consumption assay was performed in an Oxygraph apparatus equipped with a Clark-type electrode (Hansatech, U.K.).…”

“…Following by filtration using centrifugation at 8000 g (Amicon Ultra-0.5, 10 kDa) to remove the protein, the product was analyzed by HPLC on a C18 column (4.6 x 250 mm, 5-m particle size; ODS HYPERSIL) at a flow rate of 1 mL/min. The solvents used were 0.1% TFA (v/v) for buffer A and 80% acetonitrile (v/v) /0.07% TFA (v/v) for buffer B following the elution conditions reported previously [41]. The elution of product was monitored at 230 nm [29].…”