Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster

Abstract

Streptomyces lavendulae produces complestatin, a cyclic peptide natural product that antagonizes pharmacologically relevant protein-protein interactions including formation of the C4b,2b complex in the complement cascade and gp120-CD4 binding in the HIV life cycle. Complestatin, a member of the vancomycin group of natural products, consists of an alpha-ketoacyl hexapeptide backbone modified by oxidative phenolic couplings and halogenations. The entire complestatin biosynthetic and regulatory gene cluster spanning ca. 50 kb was cloned and sequenced. It consisted of 16 ORFs, encoding proteins homologous to nonribosomal peptide synthetases, cytochrome P450-related oxidases, ferredoxins, nonheme halogenases, four enzymes involved in 4-hydroxyphenylglycine (Hpg) biosynthesis, transcriptional regulators, and ABC transporters. The nonribosomal peptide synthetase consisted of a priming module, six extending modules, and a terminal thioesterase; their arrangement and domain content was entirely consistent with functions required for the biosynthesis of a heptapeptide or alpha-ketoacyl hexapeptide backbone. Two oxidase genes were proposed to be responsible for the construction of the unique aryl-ether-aryl-aryl linkage on the linear heptapeptide intermediate. Hpg, 3,5-dichloro-Hpg, and 3,5-dichloro-hydroxybenzoylformate are unusual building blocks that repesent five of the seven requisite monomers in the complestatin peptide. Heterologous expression and biochemical analysis of 4-hydroxyphenylglycine transaminon confirmed its role as an aminotransferase responsible for formation of all three precursors. The close similarity but functional divergence between complestatin and chloroeremomycin biosynthetic genes also presents a unique opportunity for the construction of hybrid vancomycin-type antibiotics.

Figure 1

Vancomycin and complestatin group of natural products.

Figure 2

(A) The three cosmids containing genes involved in the biosynthesis of complestatin. The figure is not drawn to scale. For proposed functions of assigned ORFs, see Table 1. (B) Arrangement of genes encoding NRPSs in the biosynthesis of complestatin and chloroeremomycin. A, Adenylation domain; T, thiolation domain (peptidyl carrier domain); C, condensation domain; E, epimerization domain; M, methylation domain; TE, thioesterase domain; m: NRPS module. The domain assignments and boundaries were determined by sequence comparisons of known NRPS genes. The figure is not drawn to scale.

Figure 3

Biosynthetic pathway for l-hydroxyphenylglycine. The potential destination of the p-hydroxybenzoylformate (or 3,5-dichloro-p-hydroxybenzoylformate) and Hpg (or 3,5-diClHpg) are shown in the complestatin backbone.

Figure 4

Biochemical analysis of HpgT. (A) HPLC trace showing the reactants and products in the l-Hpg reaction. A is the reaction at time 0, and B is the reaction at equlibrium. (B) HPLC trace showing the reactants and products in the d-Hpg reaction. A is the reaction at time 0, and B is the reaction at equilibrium. (C) HPLC trace showing the reactants and products in the 3,5-dichloro-l-Hpg reaction. A is the reaction at time 0, and B is the reaction at equilibrium (see text for details).