Natural products (NPs), secondary metabolites of microorganisms and plants, are great starting points for pharmaceuticals due to their structural diversity. Many NPs have complex structures and are difficult to synthesize in labs, therefore finding biosynthetic routes to harness them for biosynthetic or semisynthetic approaches is an attractive avenue for further diversification of existing NPs. Nonribosomal peptides (NRPs) which often contain nonproteinogenic amino acid moieties are one of the major targets of such efforts because of the flexibility of their biosynthetic pathways. Having nonproteinogenic amino acids in molecules may result in better bioactivity, bioavailability, and biostability. Homologation, insertion of a methylene group, is one way to synthesize these nonproteinogenic amino acids, but little is known about the pathway. HphA is an enzyme in the homologation pathway of l-phenylalanine/l-tyrosine in NP biosynthesis. In l-phenylalanine homologation, it was proposed to catalyze the reaction of phenylpyruvic acid to 2-benzyl-3-hyrdoxybutanedioic acid, which is homologous to the reaction catalyzed by LeuA in l-leucine biosynthesis. Here, HphA was biochemically characterized. The HphA-catalyzed reaction was monitored by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Time course assays were conducted to determine the reaction progress. The reaction conditions were optimized by changing the pH and the temperature. HphA had the best activity at a pH of 7.5 and a temperature of 30 °C. By using the optimized condition, the kinetic parameters for HphA with natural substrates were measured by colorimetric assays using Ellman’s reagent. The information obtained in these experiments is the initial step needed for engineering NRP biosyntheses that have homologated amino acids incorporated. This is beneficial because the engineered biosynthetic pathways can be used to make novel lead compounds that could be developed as medications in the future.

Characterization of HphA in the enzymatic homologation pathway for l-phenylalanine and l-tyrosine

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