Cryptosporidium parvum long-chain fatty acid elongase.
We report the presence of a new fatty acyl coenzyme A (acyl-CoA) elongation system in Cryptosporidium and the functional characterization of the key enzyme, a single long-chain fatty acid elongase (LCE), in this parasite. This enzyme contains conserved motifs and predicted transmembrane domains characteristic to the elongase family and is placed within the ELO6 family specific for saturated substrates. CpLCE1 gene transcripts are present at all life cycle stages, but the levels are highest in free sporozoites and in stages at 36 h and 60 h postinfection that typically contain free merozoites. Immunostaining revealed localization to the outer surface of sporozoites and to the parasitophorous vacuolar membrane. Recombinant CpLCE1 displayed allosteric kinetics towards malonyl-CoA and palmitoyl-CoA and Michaelis-Menten kinetics towards NADPH. Myristoyl-CoA (C14:0) and palmitoyl-CoA (C16:0) display the highest activity when used as substrates, and only one round of elongation occurs. CpLCE1 is fairly resistant to cerulenin, an inhibitor for both type I and II fatty acid synthases (i.e., maximum inhibitions of 20.5% and 32.7% were observed when C16:0 and C14:0 were used as substrates, respectively). These observations ultimately validate the function of CpLCE1.
Fritzler
Jason M
JM
Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, 4467 TAMU, College Station, TX 77843-4467, USA.
Millership
Jason J
JJ
Zhu
Guan
G
D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002569:Cerulenin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D016785:Cryptosporidium parvum / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005057:Eukaryotic Cells / Q000201:enzymology; D000081031:Fatty Acid Elongases; D005227:Fatty Acids / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008018:Life Cycle Stages / Q000187:drug effects; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010802:Phylogeny; D021381:Protein Transport / Q000187:drug effects; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000187:drug effects; D014162:Transfection