P450-Catalyzed Tailoring Steps in Leinamycin Biosynthesis Featuring Regio- and Stereoselective Hydroxylations and Substrate Promiscuities

Abstract

Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. Both in vivo and in vitro characterization of the LNM biosynthetic machinery have established the formation of the 18-membered macrolactam backbone and the C-3 alkyl branch; the nascent product, LNM E1, of the hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS); and the generation of the thiol moiety at C-3 of LNM E1. However, the tailoring steps converting LNM E1 to LNM are still unknown. Based on gene inactivation and chemical investigation of three mutant strains, we investigated the tailoring steps catalyzed by two cytochromes P450 (P450s), LnmA and LnmZ, in LNM biosynthesis. Our studies revealed that (i) LnmA and LnmZ regio- and stereoselectively hydroxylate the C-8 and C-4′ positions, respectively, on the scaffold of LNM; (ii) both LnmA and LnmZ exhibit substrate promiscuity, resulting in multiple LNM analogs from several shunt pathways; and (iii) the C-8 and C-4′ hydroxyl groups play important roles in the cytotoxicity of LNM analogs against different cancer cell lines, shedding light on the structure−activity relationships of the LNM scaffold and the LNM-type natural products in general. These studies set the stage for future biosynthetic pathway engineering and combinatorial biosynthesis of the LNM family of natural products for structure diversity and drug discovery.

Polyketide β-branching: diversity, mechanism and selectivity

“…106 Further processing to produce the 5-membered heterocycle and oxidation throughout the molecule takes place, however, the identity and sequence of the catalytic steps have not been proven. 44,102,[107][108][109]…”

Biocatalytic role of cytochrome P450s to produce antibiotics: A review

“…Streptomyces atroolivaceus S‐140 produces leinamycin, a powerful antitumor antibiotic, and hybrid of a polyketide and a nonribosomal peptide. In the biosynthesis of leinamycin, LnmA, and LnmZ, with regio‐ and stereoselectivity, hydroxylate the C‐8 and C‐4′ positions, respectively (Kwong et al, 2018). The dihydrothiopyran ring closure process is mediated by CxnD P450 in chuangxinmycin antibiotic biosynthesis, which is isolated from the Actinoplanes tsinanensis CPCC 200056 (Shi et al, 2018).…”

Biosynthesis of DNA-Alkylating Antitumor Natural Products

“…The PLP-dependent cysteine lyase domain (SH) can catalyze the cleavage of the C-S bond to yield 172, which is cyclized to release the chain to generate LNM E1 ( Figure 22 ) [ 135 ]. Recently, Meng et al demonstrated that LnmJ-SH domain directly installed a -SSH group into the LNM polyketide scaffold via cleavage of the C-S bond linking the thiocysteine to form LNM E ( 173 ), which might be further transferred to LNM by a set of uncharacterized enzymes [ 136 , 137 ].…”