(4-Hydroxy)mandelate synthase (HmaS) and (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) are two ␣-keto acid dependent mononuclear non-heme iron enzymes that use the same substrate, (4-hydroxyphenyl)pyruvate, but exhibit two different general reactivities. HmaS performs hydrogen-atom abstraction to yield benzylic hydroxylated product (S)-(4-hydroxy)mandelate, whereas HPPD utilizes an electrophilic attack mechanism that results in aromatic hydroxylated product homogentisate. These enzymes provide a unique opportunity to directly evaluate the similarities and differences in the reaction pathways used for these two reactivities. An Fe II methodology using CD, magnetic CD, and variable-temperature, variable-field magnetic CD spectroscopies was applied to HmaS and compared with that for HPPD to evaluate the factors that affect substrate interactions at the active site and to correlate these to the different reactivities exhibited by HmaS and HPPD to the same substrate. Combined with density functional theory calculations, we found that HmaS and HPPD have similar substrate-bound complexes and that the role of the protein pocket in determining the different reactivities exhibited by these enzymes (hydrogen-atom abstraction vs. aromatic electrophilic attack) is to properly orient the substrate, allowing for ligand field geometric changes along the reaction coordinate. Elongation of the Fe IV AO bond in the transition state leads to dominant Fe III OO •؊ character, which significantly contributes to the reactivity with either the aromatic -system or the COH -bond. magnetic circular dichroism ͉ density functional calculations ͉ reaction coordinates

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Hydroxymandelate synthase (HmaS) and hydroxyphenylpyruvate dioxygenase (HppD) are non-heme iron-dependent dioxygenases, which share a common substrate and first catalytic step. The catalytic pathways then diverge to yield hydroxymandelate for secondary metabolism, or homogentisate in tyrosine catabolism. To probe the differences between these related active sites that channel a common intermediate down alternative pathways, we attempted to interconvert their activities by directed evolution. HmaS activity was readily introduced to HppD by just two amino acid changes. A parallel attempt to engineer HppD activity in HmaS was unsuccessful, suggesting that homogentisate synthesis places greater chemical and steric demands on the active site.

ORF22 from the chloroeremomycin gene cluster has been cloned, expressed and characterised as a hydroxymandelate oxidase (HmO) that is involved in the formation of both (S)-4-hydroxyphenylglycine and (S)-3,5-dihydroxyphenylglycine.

3,5-dihydroxyphenylacetate, a precursor for the non-proteinogenic amino acid 3,5-dihydroxyphenylglycine occurring in glycopeptide antibiotics, is determined to be catalysed by a type III polyketide synthase using malonyl-CoA as a starter unit.