L-amino acid ligase from Pseudomonas syringae producing tabtoxin can be used for enzymatic synthesis of various functional peptides

Abstract

Functional peptides are expected to be beneficial compounds that improve our quality of life. To address the growing need for functional peptides, we have examined peptide synthesis by using microbial enzymes. l-Amino acid ligase (Lal) catalyzes the condensation of unprotected amino acids in an ATP-dependent manner and is applicable to fermentative production. Hence, Lal is a promising enzyme to achieve cost-effective synthesis. To obtain a Lal with novel substrate specificity, we focused on the putative Lal involved in the biosynthesis of the dipeptidic phytotoxin designated tabtoxin. The tabS gene was cloned from Pseudomonas syringae NBRC14081 and overexpressed in Escherichia coli cells. The recombinant TabS protein produced showed the broadest substrate specificity of any known Lal; it detected 136 of 231 combinations of amino acid substrates when dipeptide synthesis was examined. In addition, some new substrate specificities were identified and unusual amino acids, e.g., l-pipecolic acid, hydroxy-l-proline, and β-alanine, were found to be acceptable substrates. Furthermore, kinetic analysis and monitoring of the reactions over a short time revealed that TabS showed distinct substrate selectivity at the N and C termini, which made it possible to specifically synthesize a peptide without by-products such as homopeptides and heteropeptides with the reverse sequence. TabS specifically synthesized the following functional peptides, including their precursors: l-arginyl-l-phenylalanine (antihypertensive effect; yield, 62%), l-leucyl-l-isoleucine (antidepressive effect; yield, 77%), l-glutaminyl-l-tryptophan (precursor of l-glutamyl-l-tryptophan, which has antiangiogenic activity; yield, 54%), l-leucyl-l-serine (enhances saltiness; yield, 83%), and l-glutaminyl-l-threonine (precursor of l-glutamyl-l-threonine, which enhances saltiness; yield, 96%). Furthermore, our results also provide new insights into tabtoxin biosynthesis.

Fig 1

Overview of the substrate specificity of TabS. Reaction mixtures were analyzed by LC-ESI MS. A filled circle indicates the formation of the corresponding dipeptide. The amino acids used as substrates are shown at the left and along the bottom and are identified by a one-letter code.

Fig 2

Effects of pH (A) and temperature (B) on the peptide-synthesizing activity of TabS. Reaction mixtures were incubated for 1 h, and reaction products were analyzed by HPLC. In panel A, open circles indicate the use of 100 mM sodium phosphate buffer at pH 7 to 8 and closed circles indicate the use of 100 mM Tris-HCl buffer at pH 8 to 10. The data shown are averages of three measurements.

Fig 3

Measurement of P_i production in reaction mixtures. Black bars show the result of homopeptide synthesis, in which an arbitrary amino acid (Xaa) was used as the sole substrate. White bars show the result of heteropeptide synthesis with Glu and Xaa. Gray bars show the result of heteropeptide synthesis with Xaa and Thr. BLK shows the result when the reaction mixture was prepared without amino acid substrates. The data shown are averages of three measurements. The dashed vertical lines divide the areas, showing the preferred substrates recognized at the N and C termini.