Leishmania major possesses a unique HemG-type protoporphyrinogen IX oxidase

Abstract

Leishmania major was proposed to either utilize haem from its host or partially synthesize the tetrapyrrole from host provided precursors. However, only indirect evidence was available for this partial late haem biosynthetic pathway. Here, we demonstrate that the LMJF_06_1280 gene of L. major encodes a HemG-type PPO (protoporphyrinogen IX oxidase) catalysing the oxidation of protoporphyrinogen IX to protoporphyrin IX. Interestingly, trypanosomatids are currently the only known eukaryotes possessing HemG-type enzymes. The LMJF_06_1280 gene forms a potential transcriptional unit with LMJF_06_1270 encoding CPO (coproporphyrinogen III oxidase) and with LMJF_06_1290 for a cytochrome b5. In vivo function of the L. major hemG gene was shown by the functional complementation of the Escherichia coli ΔhemG strain LG285. Restored haem formation in E. coli was observed using HPLC analyses. Purified recombinant L. major HemG revealed PPO activity in vitro using different ubiquinones and triphenyltetrazolium as electron acceptors. FMN was identified as the L. major HemG cofactor. Active site residues were found to be essential for HemG catalysis. These data in combination with the solved crystal structures of L. major CPO and the physiological proof of a ferrochelatase activity provide clear-cut evidence for a partial haem biosynthetic pathway in L. major.

Heme synthesis through the life cycle of the heme auxotrophic parasite Leishmania major

“…These mutant bacteria were not able to grow in the absence of hemin unless they expressed LmPPOX (Fig. 2C), as previously reported by Zwerschke et al (27). This functional complementation was not inhibited by 500 nM lactofen (Fig.…”

“…If essential for the parasites, they could constitute interesting drug targets because these Leishmania enzymes differ from their human counterparts due to their bacterial origin. For example, Leishmania PPOX is the only HemG-type PPOX described in eukaryotes (27). Interestingly, plant PPOX is considered to be an ideal target for the development of herbicides, and PPOX inhibitors such as the nitrophenyl ethers acifluorfen and its (ethyl)lactate ester lactofen are used in the agricultural field (28).…”

Acquisition of exogenous haem is essential for tick reproduction

“…PPOX transcripts were also found in salivary gland transcriptomes from various species of the genus Amblyomma ( Garcia et al, 2014 ; Karim and Ribeiro, 2015 ). The same partial reduction in genomic coding for haem biosynthesis has been reported for a unicellular parasite, Leishmania major , in which the intracellular amastigote form expresses an active PPOX that likely sequesters the haem precursor coproporphyrinogen III from the macrophage cytosol to complete synthesis of its endogenous haem ( Zwerschke et al, 2014 ). Another haem auxotroph, the nematode Brugia malayi , was suggested to bypass its incomplete haem biosynthetic pathway using tetrapyrrole intermediates from endosymbionts ( Wu et al, 2009 ).…”

The Heme Transport Capacity of LHR1 Determines the Extent of Virulence in Leishmania amazonensis

“…Trypanosomatid parasites, which include the human pathogen Leishmania , are heme auxotrophs that must acquire heme from the environment to sustain essential metabolic functions [ 21 , 22 ]. Eukaryotic heme transporters were only recently identified [ 41 , 42 ] and little is known about structural features of these transporters responsible for heme translocation across membranes.…”

“…Heme functions as an essential prosthetic group for many enzymes, involved in a variety of critical cellular functions [ 20 ]. Leishmania and other trypanosomatid protozoa are heme auxotrophs that lack the first five enzymes in the heme biosynthetic pathway; to survive these parasites must acquire heme from the environment [ 6 , 21 , 22 ]. L .…”