Protostadienol synthase from Aspergillus fumigatus: functional conversion into lanosterol synthase.
Oxidosqualene:protostadienol cyclase (OSPC) from the fungus Aspergillus fumigatus, catalyzes the cyclization of (3S)-2,3-oxidosqualene into protosta-17(20)Z,24-dien-3beta-ol which is the precursor of the steroidal antibiotic helvolic acid. To shed light on the structure-function relationship between OSPC and oxidosqualene:lanosterol cyclase (OSLC), we constructed an OSPC mutant in which the C-terminal residues (702)APPGGMR(708) were replaced with (702)NKSCAIS(708), as in human OSLC. As a result, the mutant no longer produced the protostadienol, but instead efficiently produced a 1:1 mixture of lanosterol and parkeol. This is the first report of the functional conversion of OSPC into OSLC, which resulted in a 14-fold decrease in the V(max)/K(M) value, whereas the binding affinity for the substrate did not change significantly. Homology modeling suggested that stabilization of the C-20 protosteryl cation by the active-site Phe701 through cation-pi interactions is important for the product outcome between protostadienol and lanosterol.
Kimura
Miki
M
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Kushiro
Tetsuo
T
Shibuya
Masaaki
M
Ebizuka
Yutaka
Y
Abe
Ikuro
I
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D003500:Cyclization; D005672:Fusidic Acid / Q000031:analogs & derivatives / Q000096:biosynthesis; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D013185:Squalene / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D014315:Triterpenes / Q000737:chemistry / Q000378:metabolism
D014315:Triterpenes; C530466:protosta-13(17),24-dien-3beta-ol; C520393:oxidosqualene; D005672:Fusidic Acid; D013185:Squalene; D019751:Intramolecular Transferases; C021924:lanosterol synthase; C011267:helvolic acid