title: The crystal structure and activity of a putative trypanosomal nucleoside phosphorylase reveal it to be a homodimeric uridine phosphorylase.
abstract: Purine nucleoside phosphorylases (PNPs) and uridine phosphorylases (UPs) are closely related enzymes involved in purine and pyrimidine salvage, respectively, which catalyze the removal of the ribosyl moiety from nucleosides so that the nucleotide base may be recycled. Parasitic protozoa generally are incapable of de novo purine biosynthesis; hence, the purine salvage pathway is of potential therapeutic interest. Information about pyrimidine biosynthesis in these organisms is much more limited. Though all seem to carry at least a subset of enzymes from each pathway, the dependency on de novo pyrimidine synthesis versus salvage varies from organism to organism and even from one growth stage to another. We have structurally and biochemically characterized a putative nucleoside phosphorylase (NP) from the pathogenic protozoan Trypanosoma brucei and find that it is a homodimeric UP. This is the first characterization of a UP from a trypanosomal source despite this activity being observed decades ago. Although this gene was broadly annotated as a putative NP, it was widely inferred to be a purine nucleoside phosphorylase. Our characterization of this trypanosomal enzyme shows that it is possible to distinguish between PNP and UP activity at the sequence level based on the absence or presence of a characteristic UP-specificity insert. We suggest that this recognizable feature may aid in proper annotation of the substrate specificity of enzymes in the NP family.
authors.affiliation: Medical Structural Genomics of Pathogenic Protozoa Consortium, University of Washington, Seattle, WA 98195, USA.
authors.forename: Eric T
authors.initials: ET
authors.lastname: Larson
authors.forename: Devaraja G
authors.initials: DG
authors.lastname: Mudeppa
authors.forename: J Robert
authors.initials: JR
authors.lastname: Gillespie
authors.forename: Natascha
authors.initials: N
authors.lastname: Mueller
authors.forename: Alberto J
authors.initials: AJ
authors.lastname: Napuli
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Arif
authors.forename: Jenni
authors.initials: J
authors.lastname: Ross
authors.forename: Tracy L
authors.initials: TL
authors.lastname: Arakaki
authors.forename: Angela
authors.initials: A
authors.lastname: Lauricella
authors.forename: George
authors.initials: G
authors.lastname: Detitta
authors.forename: Joseph
authors.initials: J
authors.lastname: Luft
authors.forename: Frank
authors.initials: F
authors.lastname: Zucker
authors.forename: Christophe L M J
authors.initials: CL
authors.lastname: Verlinde
authors.forename: Erkang
authors.initials: E
authors.lastname: Fan
authors.forename: Wesley C
authors.initials: WC
authors.lastname: Van Voorhis
authors.forename: Frederick S
authors.initials: FS
authors.lastname: Buckner
authors.forename: Pradipsinh K
authors.initials: PK
authors.lastname: Rathod
authors.forename: Wim G J
authors.initials: WG
authors.lastname: Hol
authors.forename: Ethan A
authors.initials: EA
authors.lastname: Merritt
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D017125:Genes, Protozoan; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics; D014543:Uridine Phosphorylase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D008670:Metals; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D014543:Uridine Phosphorylase