Functional role of residues involved in substrate binding of human 4-hydroxyphenylpyruvate dioxygenase. Issue 12 (25th June 2021)

• Record Type: Journal Article
• Authors: Huang, Chih-Wei; Hwang, Chi-Ching; Chang, Yung-Lung; Liu, Jen-Tzu; Wu, Sheng-Peng; Huang, Kai-Ling; Huang, Wei-min; Lee, Hwei-Jen
• Is Part Of: Biochemical journal. Volume 478:Issue 12(2021)
• Page Start: 2201
• Page End: 2215
• Publication Date: 2021-06-25
• Subjects: 4-hydroxyphenylpyruvate dioxygenase -- C-terminal helix -- hydrogen bonding network -- non-haem Fe(II)-dependent oxygenase -- structure and function of enzyme -- substrate binding pocket; Biochemistry -- Periodicals; 572
• DOI: 10.1042/BCJ20210005
• Languages: English
• ISSNs: 0264-6021

Abstract

Abstract : 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 k cat / K m of the Asn363 mutants confirmed the critical role in catalysis. Compared to the N363A mutant, the dramatic reduction in the K d 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 K d suggest that interaction coupled with the H-bond network is crucial to locate the substrate for nucleophilic reaction.