Biosynthesis of the enediyne antitumor antibiotic C-1027 involves a new branching point in chorismate metabolism

Abstract

C-1027 is an enediyne antitumor antibiotic composed of four distinct moieties: an enediyne core, a deoxy aminosugar, a beta-amino acid, and a benzoxazolinate moiety. We now show that the benzoxazolinate moiety is derived from chorismate by the sequential action of two enzymes-SgcD, a 2-amino-2-deoxyisochorismate (ADIC) synthase and SgcG, an iron-sulfur, FMN-dependent ADIC dehydrogenase-to generate 3-enolpyruvoylanthranilate (OPA), a new intermediate in chorismate metabolism. The functional elucidation and catalytic properties of each enzyme are described, including spectroscopic characterization of the products and the development of a fluorescence-based assay for kinetic analysis. SgcD joins isochorismate (IC) synthase and 4-amino-4-deoxychorismate (ADC) synthase as anthranilate synthase component I (ASI) homologues that are devoid of pyruvate lyase activity inherent in ASI; yet, in contrast to IC and ADC synthase, SgcD has retained the ability to aminate chorismate identically to that observed for ASI. The net conversion of chorismate to OPA by the tandem action of SgcD and SgcG unambiguously establishes a new branching point in chorismate metabolism.

Fig. 1.

Structure of C-1027 with four distinct structural moieties.

Fig. 2.

Metabolic branching points from chorismate. ADC, 4-amino-4-deoxychorismate; ASI, anthranilate synthase component I; IC, isochorismate; SA, salicylic acid; ADIC, 2-amino-2-deoxyisochorismate; OPA, 3-enolpyruvoylanthranilate.

Fig. 3.

Biosynthesis of the benzoxazolinate moiety of C-1027. (A) Schematic of the C-1027 genetic locus with the eight putative ORFs identified by using BLAST analysis, including the sgcD/sgcG subcluster. (B) The enzymatic steps for the conversion of chorismate to the benzoxazolinate moiety. Highlighted in bold are the two reactions characterized in this study. Glu, glutamate; α-KG, α-ketoglutarate; FMN, flavin-mononucleotide; other abbreviations are defined in Fig. 2.

Fig. 4.

HPLC analysis of SgcD and SgcG reactions. (A) HPLC profiles of chorismate incubated with boiled SgcD (I), chorismate incubated with SgcD (II), the purified SgcD product ADIC incubated with E. coli ASI (III), and anthranilate standard (IV). (B) HPLC profiles of authentic ADIC (I), ADIC incubated with SgcG (II), ADIC incubated with ASI (III), and ADIC incubated with SgcG and spiked with authentic anthranilate (IV). Open diamonds, chorismate; filled diamonds, ADIC; open inverted triangles, anthranilate; open circles, FMN; filled circles, OPA. Anth., anthranilate; other abbreviations are defined in Fig. 2.

Fig. 5.

Kinetic analysis of SgcG. (A) Fluorescence-based assay development for SgcG with an excitation at 316 nm and emission at 400 nm. Each line represents a wavelength scan at different time points ranging from 0 to 50 min. (B) Time-course analysis of the SgcG reaction after the increase in emission intensity with different enzyme concentration: filled squares, 1× SgcG; open squares, 2× SgcG; filled circles, 4× SgcG. (C) Single-substrate kinetic analysis with variable ADIC and saturating FMN. (D) Variable FMN and saturating ADIC.