Characterisation and purification of pyruvate:ferredoxin oxidoreductase from Giardia duodenalis.
The major 2-oxoacid oxidoreductase (2-OR), pyruvate:ferredoxin oxidoreductase (PFOR) from Giardia duodenalis has been purified to apparent homogeneity. A second 2-OR with a preference for alpha-ketobutyrate as substrate was identified and was removed from PFOR containing fractions during purification. Only PFOR and the second 2-OR were identified in gels of crude Giardia extracts assayed for 2-OR activity. The native form of PFOR which is membrane associated, is a homodimer of 138 kDa subunits. Pyruvate is the preferred substrate: alpha-ketobutyrate and oxaloacetate, but not phenyl-pyruvate or alpha-ketoglutarate, are decarboxylated. PFOR from Giardia is more stable than PFOR from most other organisms and purified PFOR can be stored without deterioration at -70 degrees C. Purified PFOR donates electrons to Giardia ferredoxin (Fd I) with concomitant reduction of metronidazole. However, two other Giardia ferredoxins did not accept electrons from PFOR. Consistent with the involvement of PFOR in metronidazole activation, the activity of pyruvate dependent 2-OR activity was decreased in all metronidazole-resistant lines tested but not in furazolidone-resistant lines. The presence of three different ferredoxins and two 2-ORs in Giardia suggests that a number of different electron transport pathways operate in this organism providing unusual metabolic flexibility for a eukaryote.
Townson
S M
SM
Queensland Institute of Medical Research, Bancroft Center, Brisbane, Australia.
Upcroft
J A
JA
Upcroft
P
P
D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology; D004351:Drug Resistance; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D005872:Giardia / Q000187:drug effects / Q000201:enzymology*; D007658:Ketone Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D008795:Metronidazole / Q000494:pharmacology; D008970:Molecular Weight; D050743:Pyruvate Synthase; D013053:Spectrophotometry
D000981:Antiprotozoal Agents; D008795:Metronidazole; D007658:Ketone Oxidoreductases; D050743:Pyruvate Synthase