Biosynthetic reconstitution of deoxysugar phosphoramidate metalloprotease inhibitors using an N-P-bond-forming kinase

Abstract

Phosphoramidon is a potent metalloprotease inhibitor and a widespread tool in cell biology research. It contains a dipeptide backbone that is uniquely linked to a 6-deoxysugar via a phosphoramidate bridge. Herein, we report the identification of a gene cluster for the formation of phosphoramidon and its detailed characterization. In vitro reconstitution of the biosynthesis established TalE as a phosphoramidate-forming kinase and TalC as the glycosyltransferase which installs the l-rhamnose moiety by phosphoester linkage.

Fig. 1. Selected natural products with phosphoramidate or phosphonamidate moieties.

Fig. 2. Biosynthesis of 1 and 2. (A) Organization of the talose BGC from S. mozunensis MK-23 and gene cluster table. Genes deleted in the course of this study are indicated (Δ). Purple arrows: biosynthetic genes. Green arrows: transport and regulation. (B) Proposed pathway for the biosynthesis of 1.

Fig. 3. LC-MS analysis of culture extracts for the production of 1 in Streptomyces sp. MK730-62F2/talMB01 and 2 in S. mozunensis MK-23. Extracted ion chromatograms (EICs).

Fig. 4. LC-MS analysis of enzyme reactions. Extracted ion chromatograms (EICs) for m/z 542 [M – H]^– (1), m/z 396 [M – H]^– (5) and m/z 316 [M – H]^– (4). (a) Commercial standard of 1. (b) Enzymatic assay with TalC (protein extract of ΔtalE mutant), dTDP-l-rhamnose, TalE, 4 and ATP showed conversion of 4 to 1. (c) Control assay of (b) without TalC using a protein extract of the ΔtalC mutant. (d) Control assay of (b) without dTDP-l-rhamnose. (e) Control assay: protein extract of ΔtalC mutant with 4. (f) Control assay: protein extract of ΔtalE mutant with 4. (g) Enzymatic assay of TalE with 4 and ATP showed conversion of 4 to 5. (h) Control assay of (g) without TalE. (i) Control assay of (g) without ATP. (j) Control assay of (g) without 4. Box: observed NMR correlations of 5.