Biosynthesis of L-p-hydroxyphenylglycine, a non-proteinogenic amino acid constituent of peptide antibiotics

Background: The non-proteinogenic amino acid phydroxyphenylglycine is a cru cial component of certain peptidic natural products synthesized by a nonrib osomal peptide synthetase mechanism. In particular, for the vancomycin grou p of antibiotics phydroxyphenylglycine plays a structural role in formation of the rigid conformation of the central heptapeptide aglycone in addition to being the site of glycosylation, Initial labeling studies suggested tyr osine was a precursor of phydroxyphenylglycine but the specific steps in p- hydroxyphenylglycine biosynthesis remained unknown. Recently, the sequencin g of the chloroeremomycin gene cluster from Amycolatopsis orientalis gave n ew insights into the biosynthetic pathway and allowed for the prediction of a four enzyme pathway leading to L-p-hydroxyphenylglycine from the common metabolite prephenate. Results: We have characterized three of the four proposed enzymes of the L- p-hydroxyphenylglycine biosynthetic pathway. The three enzymes are encoded by open reading frames (ORFs) 21, 22 and 17 (ORF21: [PCZA361,1, 052791, CAA 1 1761]; ORF22: [PCZA361.2, O52792, CAA11762]; ORF17, [PCZA361.25, O52815, CAA11790]), of the chloroeremomycin biosynthetic gene cluster and we show t hey have p-hydroxymandelate synthase, p-hydroxymandelate oxidase and L-p-hy droxyphenylglycine transaminase activities, respectively. Conclusions: The L-p-hydroxyphenylglycine biosynthetic pathway shown here i s proposed to be the paradigm for how this non-proteinogenic amino acid is synthesized by microorganisms incorporating it into peptidic natural produc ts. This conclusion is supported by the finding of homologs for the four L- p-hydroxyphenylpyruvate biosynthetic enzymes in four organisms known to syn thesize peptidic natural products that contain p-hydroxyphenylglycine. Thre e of the enzymes are proposed to function in a cyclic manner in vivo with L -tyrosine being both the amino donor for L-p-hydroxyphenylglycine and a sou rce of p-hydroxyphenylpyruvate, an intermediate in the biosynthetic pathway .