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.
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.
Department of Biochemistry, National Defense Medical Center, Neihu, Taipei, Taiwan.
Jang-Foung
JF
Lin
Yung-Lin
YL
Sheih
Tsu-Chung
TC
Chang
Ni-Yuan
NY
Chang
Chiung-Wen
CW
Chang
Chia-Pei
CP
Shen
Hwei-Jen
HJ
Lee
D015111:4-Hydroxyphenylpyruvate Dioxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D055162:Biocatalysis; D020134:Catalytic Domain; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D005973:Glutamine / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010667:Phenylpyruvic Acids / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D010667:Phenylpyruvic Acids; D011994:Recombinant Proteins; D005973:Glutamine; C010590:4-hydroxyphenylpyruvic acid; D015111:4-Hydroxyphenylpyruvate Dioxygenase