| Nocardia uniformis subsp. tsuyamenensis (ATCC 21806) produces a monocyclic beta-lactam antibiotic, nocardicin A. Incorporation studies indicate nocardicin A is composed of two units of the nonproteinogenic amino acid, p-hydroxyphenylglycine (PHPG), one unit of serine, and methionine. The presence of uncommon amino acids, amide bond linkages, and D-stereocenters suggests that a nonribosomal peptide synthetase system is involved in the production of nocardicin A. In order to gain insight into the biosynthesis of the antibiotic, the corresponding gene cluster was obtained by a reverse genetics approach. The noc cluster, the first such example for a monocyclic member of the beta-lactam family, contains 14 genes required for formation, resistance, and export of nocardicin A. Among these are the genes necessary for formation of the nonproteinogenic amino acid PHPG, an AdoMet transferase that attaches an aminocarboxypropyl group to the phenolic oxygen of PHPG, and a pair of nonribosomal peptide synthetases thought to be responsible for peptide formation. The nonribosomal peptide synthetases do not follow the colinearity paradigm prescribed for these systems and may catalyze unknown transformations, such as epimerization in the absence of a specific domain or possibly beta-lactam formation. Two of the three proteins involved in PHPG biosynthesis, hydroxymandelate synthase (NocF), and hydroxymandelate oxidase (NocN), were expressed and characterized. NocF catalyzed the stereospecific transformation of substrate p-hydroxyphenylpyruvate to (S)-p-hydroxymandelate, which was then oxidized by NocN to p-hydroxybenzoylformate. NocG, which is believed to transaminate p-hydroxybenzoylformate to PHPG, concomitant with the formation of p-hydroxyphenylpyruvate from the amino donor L-tyrosine, was refractory to numerous attempts to obtain soluble, active enzyme. Finally, engineering of an enzyme with strong homology to NocF yielded unexpected insight into the mechanism of a well-studied reaction. p-Hydroxyphenylpyruvate dioxgenase catalyzes oxidative decarboxylation of the p-hydroxyphenylpyruvate side-chain, epoxidation of the aromatic ring, and a 1,2-migration of the carboxymethyl side chain in a classic example of the NIH shift to generate homogentisate. Site-directed mutagenesis of the enzyme active site to one resembling NocF and other hydroxymandelate synthases in general gave an unexpected oxepinone product, which provided the first structural evidence for the long-hypothesized arene-oxide intermediate in normal catalysis. |
