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The Biosynthesis of the Indolic Acid Moiety from the Antibiotic Nosiheptide
Author(s) -
Badding Edward,
Booker Squire
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1101.2
Subject(s) - moiety , chemistry , adenylylation , biosynthesis , stereochemistry , enzyme , isothermal titration calorimetry , biochemistry , tryptophan , amino acid , farnesyl pyrophosphate
Nosiheptide (NOS) is a highly modified thiopeptide natural product produced by Streptomyces actuosus that belongs to a unique class of thiazole‐containing peptide antibiotics. Recent studies have shown that NOS is as an effective antibacterial agent against a number of pathogenic bacteria, such as methicillin resistant Staphylococcus aureus , penicillin‐resistant Streptococcus pneumoniae , and vancomycin‐resistant enterococci. One key structural feature of NOS essential for its antibacterial properties is an indolic acid ring system characteristic of e‐series thiopeptides. The indolic acid moiety is derived from the amino acid tryptophan. In the first step of the proposed pathway, catalyzed by NosL, tryptophan is converted into 3‐methyl indolic acid (MIA). The remaining steps and enzymes involved in the formation and installation of MIA into the NOS core have not yet been definitively determined, but are believed to be catalyzed by the action of four enzymes: NosI, NosJ, NosK, and NosN. The research herein uses isothermal titration calorimetry (ITC), high‐pressure liquid chromatography (HPLC), mass spectrometry, and bioinformatics to delineate the exact roles of each of the enzymes in the biosynthesis of the indolic acid moiety. We show that NosJ is an acyl carrier protein that is post‐translationally modified with a 4′‐phosphopantetheine group, and that NosI catalyzes the ATP‐dependent adenylation of MIA. Upon incubation of NosI with ATP and MIA in the presence of NosJ, we observe transfer of MIA to NosJ. We believe that NosK then catalyzes the transfer of MIA from NosJ to the side‐chain of a cysteinyl residue of NOS. NosN, annotated as a radical S ‐adenosylmethionine (SAM) methylase, subsequently attaches a C1 unit—suggested to be a methyl group—to C4 of the indolic acid moiety and then connects the C1 unit to Glu 43 of the macrocyclic core, completing the synthesis of NOS. We are particularly interested in understanding how NosN carries out this reaction. Preliminary ITC experiments show that two SAM molecules bind to the enzyme simultaneously. We suggest that one SAM molecule is converted into a 5′‐deoxyadenosyl 5′‐radical that initiates a novel methylation reaction by abstracting a hydrogen atom from the methyl moiety of the second SAM molecule. These studies shed new light on novel mechanisms for methylating unactivated carbon centers. Support or Funding Information This work was supported by NIH (GM103268), the President's Undergraduate Research Fund (Penn State), and the Rodney Erickson Discovery Grant (Penn State).