title: Cryptosporidium parvum spermidine/spermine N1-acetyltransferase exhibits different characteristics from the host enzyme.
abstract: Cryptosporidosis is a severe opportunistic infection of immuno-compromised individuals for which no reliable therapy exists. The parasite scavenges host-derived polyamines, particularly spermine, which is then converted to the lower polyamines by the combined action of spermidine/spermine N(1)-acetyltransferase (SSAT) and polyamine oxidase (PAO). We have isolated and expressed the Cryptosporidium parvum SSAT for kinetic and molecular comparison with the host enzyme. The CpSSAT is a homotetramer with a subunit molecular mass of 18 kDa and low sequence similarity to higher eukaryotes but maintains the critical arginine residues in the active site. The CpSSAT had an activity of 299 nmol(-1)min(-1)(mg of protein)(-1) and exhibits an ordered Bi-Bi kinetics with preferred substrate specificity for spermine. Polyamine analogues having unsaturated central carbons were found to exhibit mixed inhibition kinetics of the CpSSAT. The cis-analogues were more effective inhibitors of the CpSSAT with lower K(i) values than the trans-analogues. Experiments aimed at determining the ratio of the time of the analogue in the enzyme active site to that spent out (in-out time: delta ln E/deltat) confirmed the higher efficiency of the cis-analogues as inhibitors of the CpSSAT. The results of this study reveal that the C. parvum SSAT may provide a rational target for drug design.
authors.affiliation: Haskins Laboratories, Pace University, New York, NY 10038, USA. nyarlett@pace.edu
authors.forename: Nigel
authors.initials: N
authors.lastname: Yarlett
authors.forename: Gang
authors.initials: G
authors.lastname: Wu
authors.forename: W Ray
authors.initials: WR
authors.lastname: Waters
authors.forename: James A
authors.initials: JA
authors.lastname: Harp
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Wannemuehler
authors.forename: Mary
authors.initials: M
authors.lastname: Morada
authors.forename: Demos
authors.initials: D
authors.lastname: Athanasopoulos
authors.forename: Martha P
authors.initials: MP
authors.lastname: Martinez
authors.forename: Steve J
authors.initials: SJ
authors.lastname: Upton
authors.forename: Laurence J
authors.initials: LJ
authors.lastname: Marton
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Frydman
mesh_terms: D000123:Acetyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003457:Cryptosporidiosis / Q000378:metabolism; D016785:Cryptosporidium parvum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009865:Oocytes / Q000378:metabolism; D011073:Polyamines / Q000138:chemical synthesis / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D000099228:Diamine N-Acetyltransferase
chemical_list: D000123:Acetyltransferases; D011073:Polyamines; D011994:Recombinant Proteins; D000099228:Diamine N-Acetyltransferase