Expanding the Use of Tyrosine Phenol Lyase in the Enzymatic Synthesis of Unnatural Amino Acids

Abstract

The use of enzymes in the synthesis of pharmaceuticals and biochemical tools offers an attractive approach to generating complex structures, as enzymatic reactions often lead to the formation of specific products in high yields. Tyrosine phenol lyase (TPL), an enzyme biologically involved in the degradation of tyrosine, has been used to synthesize a variety of substituted tyrosines including fluorotyrosines for biochemical studies. In addition to fluorotyrosines, expanding the scope of substrates to naphthol‐based compounds offers an attractive target for enzymatic synthesis of complex molecules, as substituted naphthols can be used as biochemical probes and as building blocks in potential pharmaceuticals. Using Citrobacter freundii TPL we enzymatically synthesized the naphthol‐analog of tyrosine, but the unnatural amino acid was generated in low yield. The simplest model for the low conversion is that the larger substrate is poorly accommodated by the enzyme active site and this poor binding leads to the low yield in the enzymatic reaction. To test this hypothesis, we mutated several bulky residues near the substrate‐binding site (M288, M397, F448) to open space for substrate binding and facilitate synthesis of the unnatural amino acid. A series of mutant plasmids were generated using site‐directed mutagenesis and sequenced to confirm the mutation. The wild type and mutant enzymes were recombinantly expressed in E. coli, purified by affinity and ion‐exchange chromatography, and purity was analyzed by gel electrophoresis. Enzyme activity with tyrosine in an NADH‐dependent coupled enzyme was first used to determine that the wild type and mutants are active, and initial results for the first mutants generated (M228S and M379A) indicate that the mutants are active. We are currently surveying reaction conditions using high‐performance liquid chromatography (HPLC) to evaluate the reactions of the TPL mutants with naphthols as substrates. HPLC is being used to determine the generation of product to calculate yields and provides a method to test if additional products are formed. While a goal of this study is aimed at the more efficient production of tyrosine derivatives, a larger goal is to better understand the features important for substrate binding in enzymatic reactions. This understanding may aid in the development of enzymes with a broad range of substrates efficiently converted to products in the biochemical generation of complex molecules.Support or Funding InformationFunding by NIH/NIGMS T34GM008074