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 alpha-ketoglutarate-dependent enzymes that couples oxidative decarboxylation of an alpha-ketoacid cofactor to oxidative modification of its substrate. In this case, the alpha-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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Gunsior M,Ravel J,Challis GL,Townsend CAdoi
10.1021/bi035762wsubject
Has Abstractpub_date
2004-01-27 00:00:00pages
663-74issue
3eissn
0006-2960issn
1520-4995journal_volume
43pub_type
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