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.
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.
Tang
Gong-Li
GL
Division of Pharmaceutical Sciences, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705, USA.
Cheng
Yi-Qiang
YQ
Shen
Ben
B
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial / Q000502:physiology; D007769:Lactams / Q000378:metabolism*; D018942:Macrolides / Q000378:metabolism*; D005810:Multigene Family / Q000502:physiology; D046908:Peptide Biosynthesis, Nucleic Acid-Independent / Q000502:physiology*; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D048630:Polyketide Synthases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013844:Thiazoles / Q000378:metabolism*; D013871:Thiones / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D007769:Lactams; D018942:Macrolides; D013844:Thiazoles; D013871:Thiones; C059000:leinamycin; D048630:Polyketide Synthases; D010453:Peptide Synthases; D000409:Alanine