Crystallographic studies of enzymes involved in biosynthesis of fatty acids : Beta-ketoacyl acyl carrier protein synthase II and delta9-stearoyl-ACP desaturase

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Medical Biochemistry and Biophysics

Sammanfattning: In the biosynthesis of fatty acids, the beta-Ketoacyl Acyl Carrier Protein Synthases (Kas) catalyse chain elongation by the addition of two-carbon units derived from malonyl-ACP, to an acyl group bound to CoA or the small protein ACP. These enzymes are potential targets for antibiotics against bacterial or parasitical infections and their mammalian counterparts could hopefully be used as drug targets against certain cancers and obesity. The crystal structure of the Kas II isoform from E. coli in complex with the well known natural inhibitor cerulenin has been determined to define the active site pocket and enable rational drug design with this target. Kas belongs to a family known as condensing enzymes for their ability to catalyse the Claisen condensation reaction. Different condensing enzymes have different substrate specificities and reaction mechanisms that somehow must be reflected in their 3D structure. The crystal structure of Kas II from Synechocystis was determined and compared to other condensing enzymes, to elucidate structural determinants for substrate specificity and reaction mechanism in condensing enzymes. Stearoyl Acyl Carrier Protein Desaturase is a soluble enzyme that catalyses the formation of a cis-double bond between carbon-9 and carbon-10 in stearic acid. Desaturase is known to contain a bent hydrophobic cavity, where the fatty acid substrate binds in the vicinity of a di-iron centre that is used for activation of molecular oxygen. Following reduction by ferredoxin, the di-iron centre activate molecular oxygen and reactive oxygen intermediates are formed that abstract hydrogen atoms from the bound fatty acid. The crystal structures of the molecular oxygen analogues, azide and acetate, in complex with desaturase have been determined in order to elucidate how the di-iron centre activates molecular oxygen and further controls the reactive oxygen intermediates to catalyse fatty acid desaturation. Crystal structures of iron depleted desaturase species have also been determined. Soluble desaturases act as the primary determinant for the composition of unsaturated fatty acids in plant membranes and seed storage oils, and variation in regio-specificity (double bond insertion position) among desaturase isozymes in certain plants is associated with the presence of unusual fatty acids in the seeds of these plants. The crystal structure of the protein complex between desaturase from Ricinus Communis and stearoyl-ACP from spinach, provides a basis for rational design of regio-specificity in soluble desaturases.

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