In silico translation and analysis of L. major genome for NUDIX domain showed that the parasite has a potential to code for at least nine putative NUDIX domain box proteins (Supplementary Table 1). The protein encoded by LmjF.31.2950 (abbreviated as ‘LmNH’ here) formed a root of the phyletic tree of these nine proteins. It also formed a single member clade in the tree as compared to remaining eight L. major NUDIX domain proteins (Supplementary Fig. 1). These two phylogenetic characters indicated that gene product of LmjF.31.2950 is a distinct and unique NUDIX protein in L. major and demanded characterization of the LmjF.31.2950 gene product. LmNH also showed presence of a highly conserved NUDIX motif that was similar to NUDIX proteins from different phyla including bacteria, plants and animals (Supplementary Table 2).
The gene LmjF.31.2950 was cloned after PCR amplification using genomic DNA isolated from L. major (ATCC 50122). The amino acid sequence of LmNH deduced from the DNA sequence of cloned LmjF.31.2950 encoding putative NUDIX hydrolase showed presence of a conserved 23 amino acid NUDIX domain box which is linked to the catalytic activity of this enzyme. LmNH also showed presence of highly conserved ‘zinc finger NADH pyrophosphatase’ (zf-NADH-PPase) domain. This domain is present in enzymes that hydrolyze reduced nicotinamide adenine dinucleotide phosphate (NAD[P]H) to AMP and reduced nicotinamide mononucleotide (NMNH). These enzymes are also known to cleave diadenosine diphosphate to AMP. Presence of a conserved amino acid sequence ‘SQPWPFPxS’ to the C-terminal side of the NUDIX domain of LmNH indicates its binding specificity to NADH, thus providing an additional evidence of its NADH pyrophosphatase activity [14]. A comparison of ‘zf-NADH-PPase’ domain of LmNH with other organisms showed that it is more homologous to bacterial enzymes than the NADH-PPase of eukaryotic species (Supplementary Table 3). Glycosomes and peroxisomes are probably derived from an ancestral peroxisome in the last eukaryotic common ancestor, however a significant number of its proteins including NUDIX proteins have an alpha-proteobacterial origin [15]. Hence, LmNH may show more homology to bacterial enzymes. It is already known that in NUDIX enzymes act in independent pathways and their substrate specificity is governed by different motifs in the protein structure and not the conserved NUDIX box [16]. Two domains involved in NAD(P)H binding namely ‘zf-NADH-PPase’ and ‘SQPWPFPxS’ were not found in Trypanosoma polyphosphate hydrolyzing enzymes.
The phyletic analysis of NUDIX hydrolases from different phyla showed that LmNH clustered in a clade that includes hydrolases localized in mitochondria and peroxisomal microbody organelles (Supplementary Fig. 2). LmNH had a canonical tri-peptide ‘SQL’ at the C-terminal end, which is a well characterized glycosomes targeting PTS-1 sequence [17] (Supplementary Table 4).
The in-depth proteomic analysis of promastigote stages of L. donovani and L. major [18, 19] showed the presence of peptides that belong to NUDIX hydrolases. The proteomic analysis of sucrose density gradient purified glycosomes [20] from L. major (unpublished data) showed a conserved peptide, ‘TGENIFSSMADNLAGR’ (MS/MS spectrum is as shown in Supplementary Fig. 3), with complete homology to LmNH amino acid sequence. To confirm the intracellular localization of this putative LmNH, the open reading frame of LmjF.31.2950 was cloned in Leishmania expression vector [21] to generate chimeric green fluorescent protein (GFP) with LmNH gene sequence at the C-terminal end of GFP. L. major expressing this chimeric reporter protein, showed a typical punctate pattern indicating its localization to microbody organelles (Fig. 1).While, cells transfected with the vector without LmNH showed a diffuse cytoplasmic fluorescence with no clear subcellular localization (data not shown). This result confirmed that LmNH is glycosomally located and targeted to glycosomes by the C-terminal PTS-1 sequence, ‘-SQL’.
In vivo verification of LmNH housecleaning function (elimination of the oxidized nucleotides) was carried out by introducing LmjF.31.2950 into an E. coli strain that is defective in mutT gene. MutT protein hydrolyzes the 8-oxodeoxyguanosine triphosphates (8-oxoGTP, known for mispairing with adenine bases during DNA replication) to 8-oxo-deoxyguanosine monophosphates [22]. E. coli CC101 is the test strain for A:T→C:G transversion, and it has an amber stop codon (UAG) at position 461 of the lacZ gene (Fig. 2A-ii). The mutT mutant of E. coli CC101T is a defined mutator strain with an increased spontaneous occurrence of unidirectional A:T→C:G transversion mutation. In this system the A:T to C:G transversion mutation leads to synthesize β-galactosidase (Fig. 2A-iii) [22]. E. coli CC101T cells alone or transformed with empty vector tend to accumulate 8-oxoGTP that increases the frequency of A:T→C:G conversion. Under these conditions, an increase in the synthesis of β-galactosidase was seen with blue color development on solid and liquid media in the presence of chromogenic substrate X-gal (Fig. 2B, c). However, E. coli CC101T cells transformed with plasmid encoding LmNH did not show any blue color development as LmNH positively complements the mutT gene function, wherein 8-oxoGTP is hydrolyzed. In such a case, the A:T→C:G conversion occurred spontaneously leading to suppressed β-galactosidase synthesis (Fig. 2B, d), proving that LmNH is complimenting the house-cleaning function of the mutated mutT gene.
Leishmania is a digenetic parasite with distinct metabolic differences in two different life cycle stages. It exists as an extracellular flagellated promastigote in the insect vector midgut. While in the mammalian host, it is present as non-motile amastigotes in the phagolysosomal compartment of macrophages. Many metabolic pathways functional in glycosomes of Leishmania are similar to those of peroxisomes in higher animals, including the pathway for carrying out β-oxidation of fatty acids. The energy requirement of the amastigote stage of the parasite is met by the increased β-oxidation of fatty acids [23]. The reducing power generated during fatty acid oxidations is in the form of NADH which remains within the microbody organelle. NADH pyrophosphatase enzyme belonging to the NUDIX superfamily catalyzes the cleavage of NADH into reduced NMP and AMP. Since the organelle membrane is believed to be impermeable to small cofactors including NADH [24, 25], hydrolysis of NADH by LmNH may be a means for regulating the quantity of the nicotinamide coenzymes within glycosomes.
Leishmania amastigotes survive and proliferate within phagolysosome that has an oxidative milieu. Oxidative stress is known to augment concentrations of the substrates of NUDIX hydrolases like di-adenosine tetraphosphate, ADP-ribose and 8-oxodGTP. In addition, glycosomes themselves have pathways that generate oxygen radicals and accumulate nonfunctional oxidized cofactors [26]. These oxidized cofactors are deleterious and their detoxification and recycling is probably carried out by housecleaning enzymes like LmNH. As purine salvage pathway and the pyrimidine biosynthetic pathways also occur within the glycosomal matrix, it is easier to predict the role of LmNH in glycosomal nucleotide metabolism. Recently several microbial Nudix hydrolases have been established to be associated with virulence and infection [27]. In light of this, NUDIX proteins from kinetoplast parasites need further investigations to elucidate its localization and function.