15N- and 13C-labeled media from Anabaena sp. for universal isotopic labeling of bacteriocins: NMR resonance assignments of leucocin A from Leuconostoc gelidum and nisin A from Lactococcus lactis.
A procedure for universal 13C and/or 15N labeling of microbial peptides which are produced by fermentation in complex media and its application to two food-preserving bacteriocins from lactic acid bacteria are described. Isotopic enrichment of nisin A (from Lactococcus lactis) and of leucocin A (from Leuconostoc gelidum) is readily achieved using a soluble peptone derived from enzymatic hydrolysis (pepsin and chymopapain) of Anabaena sp. ATCC 27899 cells grown on sodium [13C]bicarbonate and/or sodium [15N]nitrate as sole carbon and nitrogen sources. Combustion of this peptone followed by mass spectrometric analysis indicates that 45% of the labeled carbon and 65% of the labeled nitrogen added to the Anabaena culture are utilized in the amino acids of the peptone and that the isotopic purity for both 13C and 15N remains essentially unchanged provided that the cells are grown under argon atmosphere to avoid nitrogen fixation. NMR analyses of [13C,15N]nisin A using H[13C]MQC, H[13C]MBC, 2D INADEQUATE, and H[15N]MQC techniques confirmed 1H spectral assignments previously reported for unlabeled material and readily provided carbon and nitrogen assignments. The results show that universal but not uniform 13C labeling occurs unless the nutrient source is completely isotopically enriched at high level (> or = 98%) because of differential levels of de novo amino acid synthesis. Application of NMR techniques such as TOCSY, DQF-COSY, NOESY, and H[13C]MQC to unlabeled and [13C]leucocin A afforded the complete 1H and 13C assignment. Leucocin A does not possess clearly defined conformational structure in DMSO or aqueous solutions.
Sailer
M
M
Department of Chemistry, University of Alberta, Edmonton, Canada.
Helms
G L
GL
Henkel
T
T
Niemczura
W P
WP
Stiles
M E
ME
Vederas
J C
JC
D000595:Amino Acid Sequence; D017033:Anabaena / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D001430:Bacteriocins / Q000737:chemistry*; D002247:Carbon Isotopes; D007553:Isotope Labeling; D013294:Lactococcus lactis / Q000378:metabolism; D007934:Leuconostoc / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009561:Nisin / Q000737:chemistry*; D009587:Nitrogen Isotopes; D010461:Peptones / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D010461:Peptones; D009561:Nisin; C071726:leucocin A-UAL 187