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

Background: The non-proteinogenic amino acid phydroxyphenylglycine is a crucial component of certain peptidic natural products synthesized by a nonribosomal peptide synthetase mechanism. In particular, for the vancomycin group 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 tyrosine was a precursor of phydroxyphenylglycine but the specific steps in p-hydroxyphenylglycine biosynthesis remained unknown. Recently, the sequencing of the chloroeremomycin gene cluster from Amycolatopsis orientalis gave new 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, CAA1 1761]; ORF22: [PCZA361.2, O52792, CAA11762]; ORF17, [PCZA361.25, O52815, CAA11790]), of the chloroeremomycin biosynthetic gene cluster and we show they have p-hydroxymandelate synthase, p-hydroxymandelate oxidase and L-p-hydroxyphenylglycine transaminase activities, respectively. Conclusions: The L-p-hydroxyphenylglycine biosynthetic pathway shown here is proposed to be the paradigm for how this non-proteinogenic amino acid is synthesized by microorganisms incorporating it into peptidic natural products. This conclusion is supported by the finding of homologs for the four L-p-hydroxyphenylpyruvate biosynthetic enzymes in four organisms known to synthesize peptidic natural products that contain p-hydroxyphenylglycine. Three 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 source of p-hydroxyphenylpyruvate, an intermediate in the biosynthetic pathway.