Crystal structure of SgcJ, an NTF2-like superfamily protein involved in biosynthesis of the nine-membered enediyne antitumor antibiotic C-1027

Abstract

Comparative analysis of the enediyne biosynthetic gene clusters revealed sets of conserved genes serving as outstanding candidates for the enediyne core. Here we report the crystal structures of SgcJ and its homologue NCS-Orf16, together with gene inactivation and site-directed mutagenesis studies, to gain insight into enediyne core biosynthesis. Gene inactivation in vivo establishes that SgcJ is required for C-1027 production in Streptomyces globisporus. SgcJ and NCS-Orf16 share a common structure with the nuclear transport factor 2-like superfamily of proteins, featuring a putative substrate binding or catalytic active site. Site-directed mutagenesis of the conserved residues lining this site allowed us to propose that SgcJ and its homologues may play a catalytic role in transforming the linear polyene intermediate, along with other enediyne polyketide synthase-associated enzymes, into an enzyme-sequestered enediyne core intermediate. These findings will help formulate hypotheses and design experiments to ascertain the function of SgcJ and its homologues in nine-membered enediyne core biosynthesis.

Figure 1

Structures of the 9-membered enediyne natural products and their biosynthetic gene clusters. (a) Structures of the five 9-membered enediyne natural products (NCS, C-1027, KED, MDP, N1999A2) and the four additional natural products (fijiolide, SPO, CYA, CYN) proposed to be derived from 9-membered enediyne precursors after cycloaromatization. The 9-membered enediyne cores or their aromatized products are highlighted in red. (b) Alignment of the seven known 9-membered enediyne biosynthetic gene clusters highlighting the enediyne PKS gene cassette (i.e., E3, E4, E5, E, E10) (shown in red) and the seven additional conserved genes (i.e., E2, E7, E8, E9, E11, M, J) (color-coded). C-1027 biosynthetic gene cluster nomenclature is used. SgcJ and its homologues reported in this study are shown in blue and highlighted with blue boxes. Additional SgcJ homologues were also noted in the CYA and CYN clusters (boxed with dotted blue lines), but they were not included in the current study due to their varying length and significantly lower amino acid sequence homology.

Figure 2

C-1027 production by S. globisporus SB1022 and derived recombinant strains. HPLC analysis for C-1027 production from fermentation of (I) SB1022, (II) SB1027, (III) SB1028, (IV) SB1029, (V) SB1030, (VI) SB1031, (VII) SB1032, (VIII) SB1033, (IX) SB1034, and (x) SB1035. Symbols denote C-1027 chromophore (◆) and aromatized C-1027 (○). BIA (inserts) showed that the production of C-1027 in SB1022, loss of C-1027 production in SB1027, and restored C-1027 production in SB1028.

Figure 3

Sequence alignment of SgcJ and its homologues and overall structure of SgcJ and NCS-Orf16. (a) Sequence alignment of SgcJ and its homologues from the seven known 9-membered enediyne biosynthetic gene clusters. Aligned residues are colored on the basis of the level of conservation. Yellow background with red character and cyan background with red character show the putative general base and acid, respectively. Red background with white character shows strict identity, red character similarity and blue frame similarity across groups. The alignment was created with MUSCLE and rendered with ESPript 3.0. (b) Ribbon diagram and structural alignment of the SgcJ and NCS-Orf16 monomers. SgcJ and NCS-Orf16 are shown in light blue and yellow, respectively. (c) Ribbon diagram of the SgcJ dimer. The two chains are colored in blue and light blue, respectively. The NCBI accession numbers for each of the proteins are: SgcJ (ALU98438), NCS-Orf16 (AAM77985), MdpJ (ABY66022), KedJ (AFV52149), SpoJ (WP028564083), CyaJ (AG0972300), and CynJ (AG097162).

Figure 4

Structure comparison between SgcJ and selected homologous in the NTF2-like superfamily. (a) Each structure shows a curved antiparallel β-sheet wall with a group of α-helices on one side of the wall to form the cone-like shapes. The putative general base and acid are shown as yellow and green sticks, respectively. The water molecule in the structure of mgSD is depicted by a red dot. Given in parentheses are PDB accession codes for each of the structures. (b) The proposed mechanisms for KSI, mgSD, SnoaL, and Tcm ARO/CYC, featuring the conserved general acid-base pairs to catalyze the initial steps of reactions.

Figure 5

Structure comparison between SgcJ and NCS-Orf16. The electrostatics diagrams (left) and the conserved residues around the cavities (right) of (a) SgcJ and (b) NCS-Orf16. The cavities are shown by grey transparency. The ligand tetraethylene glycol (PG4) molecule is colored in green. The 2F_o – F_c electron density map is contoured at 1.0.