Chain Initiation in the Leinamycin-producing Hybrid Nonribosomal Peptide/Polyketide Synthetase from Streptomyces atroolivaceus S-140 DISCRETE, MONOFUNCTIONAL ADENYLATION ENZYME AND PEPTIDYL CARRIER PROTEIN THAT DIRECTLY LOAD

Nonribosomal peptide natural products are biosynthesized from amino acid precursors by nonribosomal peptide synthetases (NRPSs), which are organized into modules. For a typical NRPS initiationmodule, an adenylation (A) domain activates an amino acid and installs it onto a peptidyl carrier protein (PCP) domain as a thioester; an elongation module, which has a condensation (C) domain located between every consecutive pair of AandPCPdomains, catalyzes the formationof thepeptide bond between the upstream aminoacyl/peptidyl-S-PCP and the free amino group of the downstream aminoacyl-S-PCP. D-Amino acid constituents in peptide natural products usually arise from the L-enantiomers through the action of integral epimerization (E) domains of anNRPS. The biosynthetic gene cluster for leinamycin, a hybrid nonribosomal peptide/polyketide containing a D-alanine moiety, does not encode a typical NRPS initiation modulewith the expectedA-PCP-Edomains; instead, it has only an A protein (LnmQ) and a PCP (LnmP), both of which are encoded by separate genes. Here we show the results of biochemical experiments as follows: (i) we demonstrate that LnmQ directly activates D-alanine as D-alaninyl-AMP and installs it onto LnmP to generate a D-alaninyl-S-PCP intermediate; (ii) we confirm that aminoacylation of LnmP by LnmQ in trans is the result of specific communication between the separate A and PCPproteins; and (iii) we reveal that leinamycin production can be improved by supplementation of exogenous D-alanine in the fermentationbroth of Streptomyces atroolivaceousS-140.These findings unveil an unprecedented NRPS initiation module structure that is characterized by a discrete D-alanine-specific A protein and a PCP.