Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate.
The protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP+ and the inhibitor determined at 2.2 A resolution. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic centre are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the beta6-alpha6 loop and alpha6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chemical properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis.
Dawson
Alice
A
Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
Gibellini
Federica
F
Sienkiewicz
Natasha
N
Tulloch
Lindsay B
LB
Fyfe
Paul K
PK
McLuskey
Karen
K
Fairlamb
Alan H
AH
Hunter
William N
WN
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D008727:Methotrexate / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D005493:Folic Acid Antagonists; D015800:Protozoan Proteins; D010088:Oxidoreductases; C085592:pteridine reductase; D008727:Methotrexate