PMID:17502372

Citation	Tang, GL, Cheng, YQ and Shen, B (2007) 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 D-alanine. J. Biol. Chem. 282:20273-82
Abstract	Nonribosomal peptide natural products are biosynthesized from amino acid precursors by nonribosomal peptide synthetases (NRPSs), which are organized into modules. For a typical NRPS initiation module, 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 A and PCP domains, catalyzes the formation of the peptide 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 an NRPS. The biosynthetic gene cluster for leinamycin, a hybrid nonribosomal peptide/polyketide containing a D-alanine moiety, does not encode a typical NRPS initiation module with the expected A-PCP-E domains; 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 PCP proteins; and (iii) we reveal that leinamycin production can be improved by supplementation of exogenous D-alanine in the fermentation broth of Streptomyces atroolivaceous S-140. These findings unveil an unprecedented NRPS initiation module structure that is characterized by a discrete D-alanine-specific A protein and a PCP.
Links	PubMed Online version:10.1074/jbc.M702814200
Keywords	Alanine/genetics; Alanine/metabolism; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Carrier Proteins/genetics; Carrier Proteins/metabolism; Genes, Bacterial/physiology; Lactams/metabolism; Macrolides/metabolism; Multigene Family/physiology; Peptide Biosynthesis, Nucleic Acid-Independent/physiology; Peptide Synthases/genetics; Peptide Synthases/metabolism; Polyketide Synthases/genetics; Polyketide Synthases/metabolism; Protein Structure, Tertiary/physiology; Streptomyces/enzymology; Streptomyces/genetics; Thiazoles/metabolism; Thiones/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
STRAZ:Q8GGN5	involved_in	GO:0043042: amino acid adenylylation by nonribosomal peptide synthase	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
STRAZ:Q8GGN5		GO:0043042: amino acid adenylylation by nonribosomal peptide synthase	ECO:0000314:		P		Fig. 2 shows the amino acid specificity and Michaelis-Menton kinetics of LnmQ.	complete CACAO 10756
STRAZ:Q8GGN6		GO:1904091: peptidyl carrier protein activity involved in nonribosomal peptide biosynthesis	ECO:0000314:		F		Fig. 3 shows the in vitro aminoacylation of holo-LnmP by LnmQ. Fig. 4 shows the aminoacylation of LnmP by LnmQ in trans.	complete CACAO 10826
STRAZ:Q8GGN6	enables	GO:1904091: peptidyl carrier protein activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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