Abstract 1375 Enzymatic Synthesis of Naphthol-Based Unnatural Amino Acids using Tyrosine Phenol Lyase

Tyrosine derivatives are unnatural amino acids (UAAs) that are found as intermediates in the synthesis of key building blocks for anticancer compounds, such as Cryptophycin-1. Despite their importance in pharmaceutical research and production, commercial syntheses of tyrosine UAAs require multiple steps and the use of hazardous reagents, prompting a search for alternative, 'green' routes of synthesis. Tyrosine phenol lyase (TPL), isolated from Citrobacter freundii, is a pyridoxal-5′-phosphate dependent enzyme which catalyzes the reversible β-elimination of L-tyrosine into phenol, ammonium, and pyruvate. Wild-type (WT) TPL has been used previously to synthesize tyrosine UAAs, including fluorinated tyrosines starting from o-fluorophenols, but phenols with larger substituents were not efficient substrates. Multiple mutants were screened using structure-guided, site-directed mutagenesis and the M379V mutant was determined the most suitable for conversion of o-substituted phenols to 3-substituted tyrosine derivatives. In a separate study, enzyme evolution experiments identified the M288S/F448C TPL double mutant as being able to generate a two-ring 8-hydroxyquinoline analog. Although these mutations may enlarge the active site to allow larger substrate binding, the catalytic role of the specific side chains in broadening substrate scope was not investigated. To evaluate the structural basis for substrate selectivity, residues 379, 288, and 448 were mutated to valine, serine, and cysteine in single mutants, respectively. As M288S and F448C mutations also introduce polar hydrogen bonding groups, alanine mutants will also be evaluated to reduce side chain size, solely. We hypothesize that if opening space in the active site is the structural basis for accommodating the larger substrate, then the M379V and alanine mutants of residues 379, 448, and 288 should be effective at larger analog synthesis. However, if polar and hydrogen bonding capability is important, then the M379V and alanine mutants are predicted to be less effective than M288S, F488C single or double mutants. To test this hypothesis, plasmids for the mutants were generated by site-directed mutagenesis, the enzymes were expressed in E. coli, and purified by affinity chromatography followed by size-exclusion chromatography. Assays with the commonly used substrate, S-(o-nitrophenyl)-L-cysteine (SOPC) showed the enzymes were active. Next, naphthol inhibition was tested to screen if the larger substrate affects enzyme activity. The results showed greater inhibition of the M379V mutant relative to WT, suggesting that M379V increases naphthol binding. To evaluate if the enzymes generate a naphthol analog, synthesis assays were conducted and analyzed by High Performance Liquid Chromatography (HPLC). Initial HPLC results show formation of several new peaks in the chromatograms for naphthol analog synthesis reactions with the mutants, and we are currently isolating and characterizing the products formed. Together, these studies are aimed at furthering our understanding of the structural basis for broadening the substrate scope of TPL and the results may aid in using TPL to generate derivatives for use in pharmaceutical development. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number T34GM149378. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.