Engineering p-Hydroxyphenylpyruvate Dioxygenase to a p-Hydroxymandelate Synthase and Evidence for the Proposed Benzene Oxide Intermediate in Homogentisate Formation

Michele Gunsior, Jacques Ravel, Gregory L. Challis, Craig A. Townsend

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60 Citations (Scopus)

Abstract

p-Hydroxyphenylpyruvate dioxygenase (HPD) plays a key role in the normal catabolism of tyrosine. An Fe2+/oxygen-dependent enzyme, it converts p-hydroxyphenylpyruvate into homogentisate and is part of the superfamily of α-ketoglutarate-dependent enzymes that couples oxidative decarboxylation of an α-ketoacid cofactor to oxidative modification of its substrate. In this case, the α-ketoacid is part of the substrate side chain. HPD shows strong homology to p-hydroxymandelate synthase (HMS), an enzyme that catalyzes the formation of p-hydroxymandelate from p-hydroxyphenylpyruvate, an early step in the biosynthesis of p-hydroxyphenylglycine, which is a nonproteinogenic amino acid incorporated into several biologically active secondary metabolites. Sequence alignment between the HPD and the HMS enzyme families and analysis of the Pseudomonas fluorescens HPD crystal structure highlighted four residues within each active site that may play roles in catalytic differentiation between the two products. We attempted to convert Streptomyces avermitilis HPD into an engineered S. avermitilis HMS by site-directed mutagenesis of these four residues individually and in combination. HPLC assay analysis of each His6-tagged mutant indicated that F337I successfully produced p-hydroxymandelate, along with homogentisate and an unknown compound. The structure of the latter was determined to be an oxepinone derived from the benzene-oxide intermediate long hypothesized in HPD catalysis.

Original languageEnglish
Pages (from-to)663-674
Number of pages12
JournalBiochemistry
Volume43
Issue number3
DOIs
Publication statusPublished - 27 Jan 2004
Externally publishedYes

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