title: Homologues and bioengineered derivatives of LtnJ vary in ability to form D-alanine in the lantibiotic lacticin 3147.
abstract: Ltnα and Ltnβ are individual components of the two-peptide lantibiotic lacticin 3147 and are unusual in that, although ribosomally synthesized, they contain d-amino acids. These result from the dehydration of l-serine to dehydroalanine by LtnM and subsequent stereospecific hydrogenation to d-alanine by LtnJ. Homologues of LtnJ are rare but have been identified in silico in Staphylococcus aureus C55 (SacJ), Pediococcus pentosaceus FBB61 (PenN), and Nostoc punctiforme PCC73102 (NpnJ, previously called NpunJ [P. D. Cotter et al., Proc. Natl. Acad. Sci. U. S. A. 102:18584-18589, 2005]). Here, the ability of these enzymes to catalyze d-alanine formation in the lacticin 3147 system was assessed through heterologous enzyme production in a ΔltnJ mutant. PenN successfully incorporated d-alanines in both peptides, and SacJ modified Ltnα only, while NpnJ was unable to modify either peptide. Site-directed mutagenesis was also employed to identify residues of key importance in LtnJ. The most surprising outcome from these investigations was the generation of peptides by specific LtnJ mutants which exhibited less bioactivity than those generated by the ΔltnJ strain. We have established that the reduced activity of these peptides is due to the inability of the associated LtnJ enzymes to generate d-alanine residues in a stereospecific manner, resulting in the presence of both d- and l-alanines at the relevant locations in the lacticin 3147 peptides.
authors: Department of Microbiology, University College Cork, Cork, Ireland.
authors: Srinivas
authors: S
authors: Suda
authors: Elaine M
authors: EM
authors: Lawton
authors: Dorothee
authors: D
authors: Wistuba
authors: Paul D
authors: PD
authors: Cotter
authors: Colin
authors: C
authors: Hill
authors: R Paul
authors: RP
authors: Ross
mesh_terms: D000409:Alanine / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001430:Bacteriocins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D057005:Bioengineering; D013294:Lactococcus lactis / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D046937:Nostoc / Q000201:enzymology* / Q000235:genetics; D010376:Pediococcus / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D001430:Bacteriocins; D000409:Alanine