Δ9 desaturase from Trypanosoma cruzi: Key enzyme in the parasite metabolism. Cloning and overexpression

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• 作者：Mariela R. Woelke^a ; Natalia S. Paulucci^a ; Armentano Selva^a ; Hermes Garban^b ; Mirta Garcí ; a de Lema^a ; ^{mgarcia@exa.unrc.edu.ar} ; ^{mirta_b_garcia@yahoo.com}
• 关键词：FA ; fatty acids ; FID ; flame ionization detector ; FAME ; fatty acid methyl esters ; FBS ; fetal bovine serum ; IPTG ; isopropyl-β-d-thiogalactopyranoside ; SBI ; sterol biosynthesis inhibitors ; TLC ; thin layer chromatography ; PMSF ; phenylmethylsulphonyl fluoride ; LB ; Luria-Bertani ; TcΔ9DES ; Δ9 desaturase of Trypanosoma cruzi ; TcΔ9DES ; Δ9 desaturase gene of Trypanosoma cruzi
• 刊名：Microbiological Research
• 出版年：2017
• 出版时间：January 2017
• 年：2017
• 卷：194
• 期：Complete
• 页码：29-37
• 全文大小：1817 K
• 卷排序：194

文摘

Desaturases, key enzymes in the metabolism of fatty acids, regulate the physical and biochemical properties of membranes. They adjust the composition of saturated and unsaturated fatty acids in response to changes in the environmental.We demonstrated the existence of Δ9 desaturase activity in epimastigotes of the Trypanosoma cruzi Tulahuen strain. In the present study, showed that this enzyme has an approximate molecular mass of 50 kDa and a pI value of approximately 9. In order to characterize the Δ9 desaturase of Trypanosoma cruzi, (TcΔ9DES) we have cloned, sequenced and expressed in Escherichia coli. The gene consists of 1300 bp and encodes a peptide of 433 amino acids with a molecular weight of 50 kDa. Analysis of the amino acid sequence revealed three clusters of histidine and two hydrophobic regions, characteristic of membrane-bound desaturases.Gene expression studies showed that TcΔ9DES was overexpressed as an active protein. Fatty acid analysis showed that the expressed protein was confirmed to be functional with Δ9 desaturase activity. This enzyme changed the fatty acid profile of TcΔ9DES-expressing E. coli, decreasing the levels of palmitic (16:0) and stearic (18:0) acids and enhancing palmitoleic (16:1Δ9) and monounsaturated 18 carbons fatty acids. When [1–14C]palmitic or [1–14C]stearic acid was used as substrate, TcΔ9DES-expressing E. coli exhibited high desaturase activity associated with increased levels of monounsaturated fatty acids, suggesting that the TcΔ9DES enzyme was actively expressed in E. coli.To check the commitment of TcΔ9DES against sterol biosynthesis inhibitors we tested the activity under ketoconazole effect. Native TcΔ9DES, showed a significant activity inhibition.Since TcΔ9DES has shown active participation under different environmental factors, among them, ketoconazole, we consider that it plays a critical role in the metabolism of the parasite