Use of the dehydrophos biosynthetic enzymes to prepare antimicrobial analogs of alaphosphin

Abstract

The C-terminal domain of the dehydrophos biosynthetic enzyme DhpH (DhpH-C) catalyzes the condensation of Leu-tRNA^Leu with (R)-1-aminoethylphosphonate, the aminophosphonate analog of alanine called Ala(P). The product of this reaction, Leu-Ala(P), is a phosphonodipeptide, a class of compounds that have previously been investigated for use as clinical antibiotics. In this study, we show that DhpH-C is highly substrate tolerant and can condense various aminophosphonates (Gly(P), Ser(P), Val(P), 1-amino-propylphosphonate, and phenylglycine(P)) to Leu. Moreover, the enzyme is also tolerant with respect to the amino acid attached to tRNA^Leu. Using a mutant of leucyl tRNA synthetase that is deficient in its proofreading ability allowed the preparation of a series of aminoacyl-tRNA^Leu derivatives (Ile, Ala, Val, Met, norvaline, and norleucine). DhpH-C accepted these aminoacyl-tRNA derivatives and condensed the amino acid with L-Ala(P) to form the corresponding phosphonodipeptides. A subset of these peptides displayed antimicrobial activities demonstrating that the enzyme is a versatile biocatalyst for the preparation of antimicrobial peptides. We also investigated another enzyme from the dehydrophos biosynthetic pathway, the 2-oxoglutarate dependent enzyme DhpA. This enzyme oxidizes 2-hydroxyethylphosphonate to 1,2-dihydroxyethylphosphonate en route to L-Ala(P), but longer incubation results in overoxidation to 1-oxo-2-hydroxyethylphosphonate. This α-ketophosphonate was converted by the pyridoxal phosphate dependent enzyme DhpD into L-Ser(P). Thus, the dehydrophos biosynthetic enzymes can generate not only L-Ala(P) but also L-Ser(P).