Structure-function relationships of the lanthionine cyclase SpaC involved in biosynthesis of the Bacillus subtilis peptide antibiotic subtilin.
Biosynthesis of the lantibiotic subtilin in Bacillus subtilis is accomplished by a synthetase complex consisting of the dehydratase SpaB, cyclase SpaC, and transporter SpaT. Genetically engineered subtilin cyclases SpaC and related NisC and EriC proteins involved in biosynthesis of the lantibiotics nisin and ericin A/S, respectively, were analyzed to functionally substitute native SpaC in vivo. We could show for the first time posttranslational modification of a lantibiotic precursor peptide (subtilin) by a hybrid lantibiotic synthetase (SpaBT/EriC). Genetically engineered SpaC alanine replacement mutants revealed the essentiality of residues His231, Trp302, Cys303, Tyr304, Gly305, Cys349, and His350, as well as the conserved C-terminal motif Lys437-Ala438-Leu439-Leu440-Ile441 for subtilin biosynthesis. Assignment of these strictly conserved lantibiotic cyclase residues to the NisC structure [Li, B., Yu, J. B., Brunzelle, J. S., Moll, G. N., van der Donk, W. A., and Nair, S. K. (2006) Science, 311, 1464-1467] revealed the first experimental evidence for structure-function relationships in catalytic centers of lantibiotic cyclases. SpaC residues His231, Cys303, and Cys349 are involved in coordination of the central zinc ion. The pair His231/Tyr304 is discussed to act as general acid/base catalysts in lanthionine formation. Furthermore, pull-down experiments revealed that functional inactive SpaC mutants were still able to interact with the hexahistidine-tagged subtilin precursor peptide in vitro. Our results suggest that Trp302 and the C-terminal residues of SpaC are constituents of a hydrophobic cluster which is involved in stabilization of the catalytic center and binding of the subtilin precursor peptide.
Helfrich
Markus
M
Institut für Molekulare Biowissenschaften, Johann Wolfgang Goethe-Universität, Max von Laue Strasse 9, 60438 Frankfurt am Main, Germany.
Entian
Karl-Dieter
KD
Stein
Torsten
T
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001430:Bacteriocins / Q000096:biosynthesis; D004798:Enzymes / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D010455:Peptides; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D001430:Bacteriocins; D004798:Enzymes; D010455:Peptides; C002836:subtilin