Leishmania major encodes an unusual peroxidase that is a close homolog of plant ascorbate peroxidase: a novel role of the transmembrane domain

Abstract

Haem containing enzymes (peroxidase and catalase) are widely distributed among prokaryotes and eukaryotes and play a vital role in H₂O₂-detoxification. But to date no haem containing enzymatic defense against toxic-H₂O₂ has been discovered in Leishmania species. We cloned, expressed and purified an unusual plant like ascorbate peroxidase from L. major (LmAPX) and characterized its catalytic parameters under steady state condition. Examination of its protein sequence indicated approx. 30- 60% identity to other ascorbate peroxidases. The N-terminus extension of LmAPX is characterized by a charged region followed by a stretch of 22 amino acid containing a transmembrane domain. To understand how the transmembrane domain influences the structure-function of LmAPX, we generated, purified and extensively characterized a variant that lacked the transmembrane domain. Eliminating the transmembrane domain had no impact on substrate binding affinity but slowed ascorbate oxidation and increased resistance to H₂O₂ dependent inactivation in absence of electron donor by 480-folds. Spectral studies show that H₂O₂ can quickly oxidize native enzyme to compound II, which subsequently is reduced back to the native enzyme by electron donor. In contrast, ascorbate free-transmembrane domain containing enzyme did not react with H₂O₂, as revealed by absence of compound II formation. Our findings suggest that the single copy LmAPX gene may play an important role in detoxification of H₂O₂ that is generated by endogenous processes and as a result of external influences such as oxidative burst condition of infected host macrophage or during drug metabolism by Leishmania.