Premium
Probing determinants of cyclopiazonic acid sensitivity of bacterial C a 2+ ‐ ATP ases
Author(s) -
Kotšubei Aljona,
Gorgel Manuela,
Morth Jens P.,
Nissen Poul,
Andersen Jacob L.
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12310
Subject(s) - cyclopiazonic acid , lactococcus lactis , atpase , biochemistry , bacillus cereus , bacteria , biology , peptide sequence , microbiology and biotechnology , endoplasmic reticulum , lactic acid , enzyme , genetics , gene
Cyclopiazonic acid ( CPA ) is a specific and potent inhibitor of the sarcoplasmic reticulum C a 2+ ‐ ATP ase 1a ( SERCA 1a). Despite high sequence similarity to SERCA 1a, L isteria monocytogenes C a 2+ ‐ ATP ase 1 ( LMCA 1) is not inhibited by CPA . To test whether a CPA binding site could be created while maintaining the functionality of the ATP ase we targeted four amino acid positions in LMCA 1 for mutational studies based on a multiple sequence alignment of SERCA ‐like C a 2+ ‐ ATP ases and structural analysis of the CPA site. The identification of CPA ‐sensitive gain‐of‐function mutants pinpointed key determinants of the CPA binding site. The importance of these determinants was further underscored by the characterization of the CPA sensitivity of two additional bacterial C a 2+ ‐ ATP ases from Lactococcus lactis and Bacillus cereus . The CPA sensitivity was predicted from their sequence compared with the LMCA 1 results, and this was experimentally confirmed. Interestingly, a cluster of Lactococcus bacteria applied in the production of fermented cheese display C a 2+ ‐ ATP ases that are predictably CPA insensitive and may originate from their coexistence with CPA ‐producing P enicillum and A spergillus fungi in the cheese. The differences between bacterial and mammalian binding pockets encompassing the CPA site suggest that CPA derivatives that are specific for bacteria or other pathogens can be developed. Database LMCA1 ( EC: 3.6.3.8 ), SERCA1a ( EC: 3.6.3.8 )