Determining sites of interaction between prenisin and its modification enzymes NisB and NisC.
Although nisin is a model lantibiotic, our knowledge of the specific interactions of prenisin with its modification enzymes remains fragmentary. Here, we demonstrate that the nisin modification enzymes NisB and NisC can be pulled down in vitro from Lactococcus lactis by an engineered His-tagged prenisin. This approach enables us to determine important intermolecular interactions of prenisin with its modification machinery within L. lactis. We demonstrate that (i) NisB has stronger interactions with precursor nisin than NisC has, (ii) deletion of the propeptide part keeping the nisin leader intact leads to a lack of binding, (iii) NisB point mutants of highly conserved residues W616, F342A, Y346F and P639A are still able to dehydrate prenisin, (iv) NisB Δ(77-79)Y80F mutant decreased the levels of NisB-prenisin interactions and resulted in unmodified prenisin, (v) substitution of an active site residue H331A in NisC leads to higher amounts of the co-purified complex, (vi) NisB is present in the form of a dimer, and (vii) the region FNLD (-18 to -15) of the leader is an important site for binding not only to NisB, but also to NisC.
Molecular Genetics Dept., University of Groningen, Nijenborgh 7, 9747 AG Groningen, the Netherlands.
Rustem
R
Khusainov
René
R
Heils
Jacek
J
Lubelski
Gert N
GN
Moll
Oscar P
OP
Kuipers
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D013294:Lactococcus lactis / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009561:Nisin / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D014481:United States
D001426:Bacterial Proteins; D008565:Membrane Proteins; D050505:Mutant Proteins; C079126:NisB protein, Lactococcus lactis; D011498:Protein Precursors; D009561:Nisin