Intermediate Partitioning Kinetic Isotope Effects for the NIH Shift of 4-Hydroxyphenylpyruvate Dioxygenase and the Hydroxylation Reaction of Hydroxymandelate Synthase Reveal Mechanistic Complexity

Abstract

4-Hydroxyphenylpyruvate dioxygenase (HPPD) and hydroxymandelate synthase (HMS) are similar enzymes that catalyze complex dioxygenation reactions using the substrates 4-hydroxyphenylpyruvate (HPP) and dioxygen. Both enzymes decarboxylate HPP and then hydroxylate the resulting hydroxyphenylacetate (HPA). The hydroxylation reaction catalyzed by HPPD displaces the aceto substituent of HPA in a 1,2-shift to form 2,5-dihydroxyphenylacetate (homogentisate, HG), whereas the hydroxylation reaction of HMS places a hydroxyl on the benzylic carbon forming 3'-hydroxyphenylacetate (S-hydroxymandelate, HMA) without ensuing chemistry. The wild-type form of HPPD and variants of both enzymes uncouple to form both native and non-native products. We have used intermediate partitioning to probe bifurcating steps that form these products by substituting deuteriums for protiums at the benzylic position of the HPP substrate. These substitutions result in altered ratios of products that can be used to calculate kinetic isotope effects (KIE) for the formation of a specific product. For HPPD, secondary normal KIEs indicate that cleavage of the bond in the displacement reaction prior to the shift occurs by a homolytic mechanism. NMR analysis of HG derived from HPPD reacting with enantiomerically pure R-3'-deutero-HPP indicates that no rotation about the bond to the radical occurs, suggesting that collapse of the biradical intermediate is rapid. The production of HMA was observed in HMS and HPPD variant reactions. HMS hydroxylates to form exclusively S-hydroxymandelate. When HMS is reacted with R-3'-deutero-HPP, the observed kinetic isotope effect represents geometry changes in the initial transition state for the nonabstracted proton. These data show evidence of sp(3) hybridization in a HPPD variant and sp(2) hybridization in HMS variants, suggesting that HMS stabilizes a more advanced transition state in order to catalyze H-atom abstraction.

Mechanistic Insight on the Activity and Substrate Selectivity of Nonheme Iron Dioxygenases

“…One key experimental result assigned a rate‐determining hydrogen atom abstraction step in the reaction performed by the iron(IV)‐oxo intermediate, which was determined from kinetics studies by replacing the transferring hydrogen atom by deuterium. The rate constant change led to a significant kinetic isotope effect; therefore, the hydrogen atom abstraction is rate determining …”

The molecular basis for the intramolecular migration (NIH shift) of the carboxyl group during para‐hydroxybenzoate catabolism

“…To date, the NIH shift has been found not only in bacteria (Guroff et al, 1966;Buswell and Clark, 1976;Crawford, 1976;Keenan and Chapman, 1978;Hartmann et al, 1999;Gabriel et al, 2012) and Archaea (Fairley et al, 2002), but also in eukarya, including Homo sapiens (Lindstedt and Odelhög, 1987;Hu et al, 2003). The substituents subject to NIH shifts include hydrogen (deuterium or tritium) (Hartmann et al, 1999;Hu et al, 2003), halogen (Mori et al, 2009), aceto substituent (Hareland et al, 1975;Rundgren, 1982;Moran et al, 2000;Moran, 2005;Shah et al, 2013), alkyl group (Gabriel et al, 2012) and carboxyl group (Buswell and Clark, 1976;Crawford, 1976;Keenan and Chapman, 1978;Fairley et al, 2002;Schoenian et al, 2012). The NIH shift of hydrogen is found to be involved in disorder of amino acid metabolism (phenylalanine and tryptophan) (Jequier et al, 1969;Waisbren and Levy, 1991) and in metabolism of therapeutic drugs (such as tamoxifen) (Hu et al, 2003).…”

Go it alone: four-electron oxidations by mononuclear non-heme iron enzymes

“…Experimental attempts to elucidate the HPPD mechanism after generation of the putative Fe(IV)=O species have relied on indirect methods, including mutants of HPPD that uncouple oxidative decarboxylation from native product formation to instead make both the native product and shunt products. These mutated proteins were then reacted with deuterated substrates and the ratio of shunt to native products was determined to infer kinetic isotope effects (KIEs) on isotopically-sensitive branching steps [31, 32]. These experiments led to the conclusion that a homolytic biradical mechanism was responsible for the alkyl migration, consistent with early DFT calculations [33].…”