Dissecting the Metabolic Roles of Pteridine Reductase 1 in Trypanosoma brucei and Leishmania major

• DOI: 10.1074/jbc.m110.209593
• Publication Date: 2011-01-15T02:09:35Z
• Authors: Han B. Ong; Natasha Sienkiewicz; Susan Wyllie; Alan H. Fairlamb
• Keywords: Pteridine; Pterin; Biopterin; Mesoionic

Leishmania parasites are pteridine auxotrophs that use an NADPH-dependent reductase 1 (PTR1) and NADH-dependent quinonoid dihydropteridine (QDPR) to salvage maintain intracellular pools of tetrahydrobiopterin (H(4)B). However, the African trypanosome lacks a credible candidate QDPR in its genome despite maintaining apparent activity. Here we provide evidence activity previously reported by others is assay artifact. Using HPLC-based enzyme assay, demonstrate there associated with both TbPTR1 LmPTR1. The kinetic properties recombinant PTR1s at physiological pH ionic strength compared LmQDPR. Specificity constants (k(cat)/K(m)) for LmPTR1 similar dihydrobiopterin (H(2)B) (qH(2)B) as substrates about 20-fold lower than LmQDPR qH(2)B. In contrast, shows 10-fold higher k(cat)/K(m) H(2)B over Analysis Trypanosoma brucei isolated from infected rats revealed H(4)B (430 nM, 98% total biopterin) was predominant pterin, consistent dual role regeneration H(4)B. Gene knock-out experiments confirmed this: PTR1-nulls could only be obtained lines overexpressing medium supplement. These cells grew normally H(4)B, which spontaneously oxidizes qH(2)B, but were unable survive absence pterin or either biopterin medium. findings establish PTR1 has essential metabolism trypanosomes underline potential drug target.