Structural and Biochemical Characterization of the Salicylyl-acyltranferase SsfX3 from a Tetracycline Biosynthetic Pathway

Abstract

Background: SsfX3 is an acyltransferase that can acylate tetracycline-like molecules at C-4. Results: Crystal structure showed SsfX3 contains a structural N-terminal ␤-sandwich domain and a catalytic C-terminal hydrolase domain. Mutagenesis revealed both are essential for activity. Conclusion:The N-terminal domain is recruited to bind the large "small molecule" substrate. Significance: New structural knowledge enables engineering of the acyltransferase toward synthesis of tetracycline analogs.

Crystal structure of SsfS6, the putative C‐glycosyltransferase involved in SF2575 biosynthesis

“…Based upon the determined structure of the SsfS6‐TDP complex, we postulate either D58 or E316 may serve as the active‐site base and the side chain of H292 may be used to stabilize the β‐phosphate during catalysis. While the nature of the actual SsfS6 aglycon remains to be elucidated, and co‐crystallization experiments with a range of tetracycline analogs were unsuccessful, molecular docking was employed in an attempt to glean additional information . Four docked tetracycline‐like aglycon orientations were identified as potential poses in which the protein SsfS6 could productively interact [Supporting Information Fig.…”

Heterologous Expression and Manipulation of Three Tetracycline Biosynthetic Pathways

“…[11] In the oxy pathway, the C4 ketone group in 7 is reductively aminated and N,N -dimethylated to yield 5 . [15] The presence of the R -C4-hydroxyl group in 9 indicates that ketoreduction at C4 by a ketoreductase should take place early in the pathway.…”

“…[7,8] The 2-carboxamide tetracyclic structure universal to all tetracyclines has been recognized as a privileged scaffold for drug discovery. [9] We report herein the heterologous expression and genetic manipulation of three structurally diverse natural tetracyclines: the classic antibiotic oxytetracycline (1), [10] the antitumor agent SF2575 (2), [11] and dactylocycline (3), which is active against tetracycline-resistant bacterial strains. [12] Numerous new tetracycline compounds were obtained from the engineered host/pathway pairs, which led to the discovery of a new set of tailoring enzymes that can modify the tetracycline scaffold at different positions.…”

“…2575, only limited in vitro experiments have been performed on the ssf tailoring enzymes, such as the salicylate transferase SsfX3. [11] To construct a K4-114 host for the production of 2, we integrated pSF2575 encoding the entire ssf gene cluster (40 genes, 47 kb) [27] into the chromosome and overexpressed the SARP SsfT1 under the ermE* promoter to yield K4/pSF2575-T1 (see Table S1). HPLC-MS analysis showed the production of 2 and two intermediates 13 and 14 with a combined titer of approximately 127 mg L À1 (Figure 2 A and Scheme 1 B).…”

Biosynthesis of the antituberculous agent caprazamycin: Identification of caprazol-3ʺ-phosphate, an unprecedented caprazamycin-related metabolite

“…Cpz23 shows 33% sequence identity and 52% sequence similarity to SsfX3 acyltransferase, which catalyzes the C-4 acylation of salicylic acid in the tetracycline skeleton by using salicylicCoA as a cofactor (Pickens et al, 2011) (Fig. S6).…”