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Functional role of residues involved in substrate binding of human 4-hydroxyphenylpyruvate dioxygenase
Author(s) -
Chih-Wei L. Huang,
Chi-Ching Hwang,
YungLung Chang,
Jen-Tzu Liu,
Sheng-Peng Wu,
Kuo Cheng Huang,
Weimin Huang,
Hwei-Jen Lee
Publication year - 2021
Publication title -
biochemical journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.706
H-Index - 265
eISSN - 1470-8728
pISSN - 0264-6021
DOI - 10.1042/bcj20210005
Subject(s) - stereochemistry , chemistry , dioxygenase , helix (gastropod) , mutant , substrate (aquarium) , binding site , enzyme kinetics , ligand (biochemistry) , protein structure , active site , enzyme , biochemistry , biology , receptor , ecology , snail , gene
4-Hydroxylphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxylphenylpyruvate (HPP) to homogentisate, the important step for tyrosine catabolism. Comparison of the structure of human HPPD with the substrate-bound structure of A. thaliana HPPD revealed notably different orientations of the C-terminal helix. This helix performed as a closed conformation in human enzyme. Simulation revealed a different substrate-binding mode in which the carboxyl group of HPP interacted by a H-bond network formed by Gln334, Glu349 (the metal-binding ligand), and Asn363 (in the C-terminal helix). The 4-hydroxyl group of HPP interacted with Gln251 and Gln265. The relative activity and substrate-binding affinity were preserved for the Q334A mutant, implying the alternative role of Asn363 for HPP binding and catalysis. The reduction in kcat/Km of the Asn363 mutants confirmed the critical role in catalysis. Compared to the N363A mutant, the dramatic reduction in the Kd and thermal stability of the N363D mutant implies the side-chain effect in the hinge region rotation of the C-terminal helix. The activity and binding affinity were not recovered by double mutation; however, the 4-hydroxyphenylacetate intermediate formation by the uncoupled reaction of Q334N/N363Q and Q334A/N363D mutants indicated the importance of the H-bond network in the electrophilic reaction. These results highlight the functional role of the H-bond network in a closed conformation of the C-terminal helix to stabilize the bound substrate. The extremely low activity and reduction in Q251E's Kd suggest that interaction coupled with the H-bond network is crucial to locate the substrate for nucleophilic reaction.

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