Effect of the hydrophobic pocket structure on catalytic properties of theB-ketoacylACP synthases.

TRAJTENBERG F; ALTABE, SILVIA GRACIELA; FICARRA,F; BUSCHIAZZO ,ALEJANDRO; DE MENDOZA, D; SCHUJMAN, G

Tucuman

Congreso; XLV Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2009

SAIB

Effect of the hydrophobic pocket structure on catalytic properties of theB-ketoacylACP synthases.These enzymes are potently inhibited by the fungal mycotoxin cerulenine a 12C fatty acid analog cerulenine which binds covalenlty to the KAS active site thiol.We have previously describe a B. subtilis strain containing a fabF1 , which encodes for the cerulenin-insensitive protein FabF[I108F]. This strain had a cerulenin MIC 8-fold higher than the wild type strain. Although B subtilis FabF[I108F] is functional both in vivo and in vitro, the E. coli FabF[I108F] enzyme is reported to be unable to elongate medium- and long-chain acyl-ACPs.Here we report the characterization of another two spontaneous mutants of B. subtilis that exhibit 4-fold increase in the cerulenin MIC. These mutants carry the fabF2 (L111F) and fabF3 (I108M) alleles. Biochemical and genetic studies were performed to understand the different properties of wild type and mutated FabFs from B subtilis and E. coli. Also, the high resolution structure of FabF1 alone and in complex with cerulenin was obtained.These studies provide major insights into the key elements that contribute to the activity of this important class of enzymes and the mechanisms associated with the resistance to cerulenin.