title: Expression and functional characterization of a giant Type I fatty acid synthase (CpFAS1) gene from Cryptosporidium parvum.
abstract: A 25-kb CpFAS1 gene from Cryptosporidium parvum has been engineered and expressed as five individual maltose-binding protein (MBP)-fusion proteins: an N-terminal loading unit, three fatty acyl elongation modules, and a C-terminal reductase. Enzymatic activities of all domains (except the reductase) were individually assayed as recombinant proteins. The preferred substrate for the fatty acyl ligase (AL) domain in the loading unit was palmitic acid (C16:0). However, a competition assay suggests that the AL domain could also utilize other fatty acids as substrates (i.e., C12:0-C24:0), albeit with reduced activity. Among the three elongation modules, enzymatic activities were detected for ketoacyl synthase (KS), acyl transferase (AT), dehydrase (DH), enoyl reductase (ER), and ketoacyl reductase (KR) domains, which suggests that these modules were involved in the elongation of a saturated fatty acyl chain that would be C6 longer (e.g., C22:0) than the precursor (e.g., C16:0). In addition, the KS activity could be specifically inhibited by cerulenin (IC(50) approximately 1.5 microM), reinforcing the notion that CpFAS1 could be exploited as potential drug target. Since C. parvum lacks other fatty acid synthases, these observations imply that this parasite may not be capable of synthesizing fatty acids de novo.
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, 4467 TAMU, College Station, TX 77843, USA. gzhu@cvm.tamu.edu
authors.forename: Guan
authors.initials: G
authors.lastname: Zhu
authors.forename: Yanan
authors.initials: Y
authors.lastname: Li
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Cai
authors.forename: Jason J
authors.initials: JJ
authors.lastname: Millership
authors.forename: Mary J
authors.initials: MJ
authors.lastname: Marchewka
authors.forename: Janet S
authors.initials: JS
authors.lastname: Keithly
mesh_terms: D000818:Animals; D002569:Cerulenin / Q000494:pharmacology; D003001:Cloning, Molecular; D016785:Cryptosporidium parvum / Q000201:enzymology* / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D064429:Fatty Acid Synthases / Q000235:genetics* / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D017125:Genes, Protozoan; D017434:Protein Structure, Tertiary / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D005227:Fatty Acids; D011993:Recombinant Fusion Proteins; D002569:Cerulenin; D064429:Fatty Acid Synthases