Evidence for the Mechanism of Hydroxylation by 4-Hydroxyphenylpyruvate Dioxygenase and Hydroxymandelate Synthase from Intermediate Partitioning in Active Site Variants

Abstract

4-Hydroxyphenylpyruvate dioxygenase (HPPD) and hydroxymandelate synthase (HMS) each catalyze similar complex dioxygenation reactions using the substrates 4-hydroxyphenylpyruvate (HPP) and dioxygen. The reactions differ in that HPPD hydroxylates at the ring C1 and HMS at the benzylic position. The HPPD reaction is more complex in that hydroxylation at C1 instigates a 1,2-shift of an aceto substituent. Despite that multiple intermediates have been observed to accumulate in single turnover reactions of both enzymes, neither enzyme exhibits significant accumulation of the hydroxylating intermediate. In this study we employ a product analysis method based on the extents of intermediate partitioning with HPP deuterium substitutions to measure the kinetic isotope effects for hydroxylation. These data suggest that, when forming the native product homogentisate, the wild-type form of HPPD produces a ring epoxide as the immediate product of hydroxylation but that the variant HPPDs tended to also show the intermediacy of a benzylic cation for this step. Similarly, the kinetic isotope effects for the other major product observed, quinolacetic acid, showed that either pathway is possible. HMS variants show small normal kinetic isotope effects that indicate displacement of the deuteron in the hydroxylation step. The relatively small magnitude of this value argues best for a hydrogen atom abstraction/rebound mechanism. These data are the first definitive evidence for the nature of the hydroxylation reactions of HPPD and HMS.

Role of Substrate Positioning in the Catalytic Reaction of 4-Hydroxyphenylpyruvate Dioxygenase—A QM/MM Study

“…mitilis HPPD. 17,27 With ring-deuterated HPP inverse secondary KIE of 0.84 was obtained, which was interpreted as evidence for a change of hybridization from sp 2 to sp 3 on the carbon atom(s) directly bound to deuterium, i.e. direct attack of the ferryl species on the C1-C2 bond of the aromatic ring and formation of a 1,2-epoxide.…”

“…7 Similarities in sequences, structures and the reaction mechanisms of HMS and HPPD make these enzymes very attractive targets for mutagenesis experiments aimed at switching the hydroxylation specificity. [15][16][17] Results of mutagenesis studies showed that HMS is very tolerant to mutations and single, double and triple amino acid substitutions at conserved positions have not led to production of HG. 15,16 In the case of HPPD, analogous attempts were more successful and some mutants produced HMA.…”

“…These observations might indicate that the HMS reaction is the "default" oxidation process for the (2His-1Glu)-Fe(IV)=O-HPA catalytic core, presumably less controlled by interactions with the second shell residues than ring hydroxylation specific for HPPD. [15][16][17] Results of Gunsior et al showed that two HPPD (S. avermitilis) single-point mutants, namely Asn216Ile and Phe337Ile, form mixture of products including HG and HMA. 16 It was suggested that structural changes introduced by these point mutations inhibit formation of HG and hence turn on production of the side product -HMA.…”

“…direct attack of the ferryl species on the C1-C2 bond of the aromatic ring and formation of a 1,2-epoxide. 17 HPPD mutants, such as: Asn245Ile, Asn245Gln, Pro243Thr and Ser230Ala, gave secondary KIE of around 1. 17 In studies using 1',1'-dideutero-HPP the KIE value obtained for WT Streptomyces avermitilis HPPD was 0.990, which was proposed to be consistent with a heterolytic single step mechanism of carboxymethyl migration.…”

“…17 HPPD mutants, such as: Asn245Ile, Asn245Gln, Pro243Thr and Ser230Ala, gave secondary KIE of around 1. 17 In studies using 1',1'-dideutero-HPP the KIE value obtained for WT Streptomyces avermitilis HPPD was 0.990, which was proposed to be consistent with a heterolytic single step mechanism of carboxymethyl migration. 28 However, for the N216(245)S mutant KIE amounted to 1.39, a value presumably more consistent with the radical 2-step migration.…”