Biochemical characterization of a prokaryotic phenylalanine ammonia lyase

Abstract

The committed biosynthetic reaction to benzoyl-coenzyme A in the marine bacterium "Streptomyces maritimus" is carried out by the novel prokaryotic phenylalanine ammonia lyase (PAL) EncP, which converts the primary amino acid L-phenylalanine to trans-cinnamic acid. Recombinant EncP is specific for L-phenylalanine and shares many biochemical features with eukaryotic PALs, which are substantially larger proteins by approximately 200 amino acid residues.

FIG. 1.

EncP-catalyzed conversion of phenylalanine to trans-cinnamic acid and biosynthesis of benzoyl-CoA-derived enterocin in “S. maritimus.”

FIG. 2.

Relatedness tree of aromatic amino acid ammonia lyases from prokaryotes and eukaryotes. Sequences were retrieved from GenBank (accession numbers are given in parentheses) and aligned with ClustalX (1.83) using the neighbor-joining method.

FIG. 3.

(A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified, octahistidyl-tagged EncP. Lane 1, molecular size markers (kDa); lane 2, His₈-EncP (calculated molecular mass is 58.7 kDa). (B) pH dependence on the rate of trans-cinnamic acid formation from l-phenylalanine by EncP.

FIG. 4.

Assay progress curves in the presence of AIP. EncP was incubated at 40°C in 100 mM Tris-HCl (pH 8.0) containing 0.4 mM l-phenylalanine and increasing concentrations of AIP. The reaction was started by the addition of the enzyme. Points represent averaged values from duplicate experiments.