Detection of cleavage of a prenisin-mimicking decapeptide by Lactococcus lactis subsp. lactis endoproteinase activity.
As part of ongoing studies in the biosynthesis of the lantibiotic nisin, we investigated the proteolytic cleavage of a synthetic Fmoc-labeled decapeptide mimicking a key amino acid sequence of the precursor prenisin in extracts of a Lactococcus lactis subsp. lactis strain. Reverse-phase high-performance liquid chromatography with photodiode array detection was used to trace and purify potential enzymatic conversion products. Of the three newly appearing chromatographic peaks, one was identified by means of electrospray mass spectrometry, amino acid analysis, and amino acid sequencing as an Fmoc-labeled hexapeptide derived from cleavage at the Arg-1-Ile+1 bond. This assay will be useful to monitor the purification of the endoproteinase that reportedly cleaves prenisin at the same Arg-1-Ile+1 site present in the model substrate.
Nelis
H J
HJ
Laboratoria voor Medische Biochemie en voor Klinische Analyse, Universiteit Gent, Belgium.
De Vuyst
L
L
Goubert
K
K
Devreese
B V
BV
Van Beeumen
J J
JJ
Raymackers
J G
JG
Vandamme
E J
EJ
De Leenheer
A P
AP
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000032:analysis / Q000378:metabolism*; D013294:Lactococcus lactis / Q000201:enzymology / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009561:Nisin / Q000378:metabolism / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D011498:Protein Precursors / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D013053:Spectrophotometry
D000900:Anti-Bacterial Agents; D010455:Peptides; D011498:Protein Precursors; D009561:Nisin; D010450:Endopeptidases