Plasmodium Falciparum Genome Research Articles

Increased Polymorphism Near Low-Complexity Sequences across the Genomes of Plasmodium falciparum Isolates

Low-complexity regions (LCRs) within proteins sequences are often considered to evolve neutrally even though recent studies reported evidence for selection acting on some of them. Because of their widespread distribution among eukaryotes genomes and the potential deleterious effect of expansion/contraction of some of them in humans, low-complexity sequences are of major interest and numerous studies have attempted to describe their dynamic between genomes as well as the factors correlated to their variation and to assess their selective value. However, due to the scarcity of individual genomes within a species, most of the analyses so far have been performed at the species level with the implicit assumption that the variation both in composition and size within species is too small relative to the between-species divergence to affect the conclusions of the analysis. Here we used the available genomes of 14 Plasmodium falciparum isolates to assess the relationship between low-complexity sequence variation and factors such as nucleotide polymorphism across strains, sequence composition, and protein expression. We report that more than half of the 7,711 low-complexity sequences found within aligned coding sequences are variable in size among strains. Across strains, we observed an increasing density of polymorphic sites toward the LCR boundaries. This observation strongly suggests the joint effects of lowered selective constraints on low-complexity sequences and a mutagenic effect of these simple sequences.

Using increment of diversity to predict mitochondrial proteins of malaria parasite: integrating pseudo-amino acid composition and structural alphabet

Due to the complexity of Plasmodium falciparum (PF) genome, predicting mitochondrial proteins of PF is more difficult than other species. In this study, using the n-peptide composition of reduced amino acid alphabet (RAAA) obtained from structural alphabet named Protein Blocks as feature parameter, the increment of diversity (ID) is firstly developed to predict mitochondrial proteins. By choosing the 1-peptide compositions on the N-terminal regions with 20 residues as the only input vector, the prediction performance achieves 86.86% accuracy with 0.69 Mathew's correlation coefficient (MCC) by the jackknife test. Moreover, by combining with the hydropathy distribution along protein sequence and several reduced amino acid alphabets, we achieved maximum MCC 0.82 with accuracy 92% in the jackknife test by using the developed ID model. When evaluating on an independent dataset our method performs better than existing methods. The results indicate that the ID is a simple and efficient prediction method for mitochondrial proteins of malaria parasite.

A novel dual Dbp5/DDX19 homologue from Plasmodium falciparum requires Q motif for activity

Helicases are ubiquitous essential enzymes which have significant role in the nucleic acid metabolism. Using in silico approaches in the recent past we have identified a number of helicases in the Plasmodium falciparum genome. In the present study we report purification and detailed characterization of a novel helicase from P. falciparum. Our results indicate that this helicase is a homologue of Dbp5 and DDX19 from yeast and human, respectively. The biochemical characterization shows that it contains DNA and RNA unwinding, nucleic acid dependent ATPase and RNA binding activities. It is interesting to note that this enzyme can unwind DNA duplexes in both 5′ to 3′ and 3′ to 5′ directions. Using truncated derivatives we further show that Q motif is essentially required for all of its activities. These studies should make an important contribution in understanding the enzymes involved in nucleic acid metabolism in the parasite.

Recombinant expression, purification and copper-binding characteristics of the amino terminus of a Plasmodium falciparum copper transport protein

Copper is an essential micronutrient for all living organisms since it is required as a catalytic cofactor for metabolic enzymes, such as cytochrome-c oxidase. In the higher eukaryotes copper is acquired by transport across the plasma membrane via the copper transport protein, Ctr1. The malaria parasite expresses copper requiring enzymes, such as S-adenosylhomocysteine hydrolase and cytochrome-c oxidase as well as copper chaperones, such as Cox17. How the parasite acquires copper has, however, not yet been described. In silico BLAST-P screening of the Plasmodium database (www.PlasmoDB. org), using a putative copper transport protein sequence from the related parasite Theileria parva, identified a candidate copper transport protein sequence for the malaria parasite. The sequence was present in the genome of human, monkey, avian and rodent infecting species of Plasmodium. Each sequence contained features typical of copper transport proteins such as copperbinding motifs in the N-terminal region, three membrane spanning domains as well as the characteristic MxxxM and GxxxG motifs, located in the second and third transmembrane domains respectively. Two putative copper transport proteins are predicted to be present in the Plasmodium falciparum genome (PF14_0211 and PF14_0369). The predicted N-terminal domains for each protein were cloned and expressed as soluble 48kD (PF14_0211) and 54kD (PF14_0369) fusion partners with maltose-binding protein. Copper was shown to bind to the purified recombinant fusion proteins both in vivo and in vitro using the copper-specific bicinchoninic acid assay. For each protein an immunogenic peptide, within the N-terminus, was selected, synthesised, coupled to rabbit albumin carrier and used to raise antibodies in chickens. The antibodies were affinity purified and used to probe for malarial proteins. Copper is important for a range of enzyme activities in malaria parasites, which suggests that a malaria copper transport protein is an interesting target for novel anti-malarial compounds.

Evidence for prenylation-dependent targeting of a Ykt6 SNARE in Plasmodium falciparum

Ykt6 proteins are the most versatile fusogens in eukaryotic cells, and the only SNAREs that can be both prenylated and acylated at a C-terminal CAAX motif. Unlike yeast and mammalian cells where a single Ykt6 gene is expressed, the Plasmodium falciparum genome encodes two Ykt6 proteins. We have investigated the expression and prenylation of the Ykt6 orthologue, PfYkt6.1 in intra-erythrocytic stages of P. falciparum. PfYkt6.1 localized to the parasite Golgi and other unidentified cytoplasmic compartments, and was partly cytosolic (∼50% in early trophozoites). The membrane-association of PfYkt6.1 was dependent on the presence of a conserved C-terminal CAAX motif (CCSIM). By expressing full-length and mutant proteins in Escherichia coli, we have shown that PfYkt6.1 indeed serves as substrate for prenylation by P. falciparum farnesyltransferases. Surprisingly, PfYkt6.1 could also be geranylgeranylated by parasite extracts independent of the C-terminal amino acid residue. Deletion of the CAAX motif inhibited both farnesylation and geranylgeranylation activities. Additionally, the PfYkt6.1 heptapeptide KQCCSIM, corresponding to the C-terminal CAAX sequence, inhibited the parasite farnesyltransferase activity with an IC50 of 1μM. Our findings underscore the importance of CAAX motif-derived peptidomimetics for antimalarial drug development.

A Genetic Screen for Attenuated Growth Identifies Genes Crucial for Intraerythrocytic Development of Plasmodium falciparum

A majority of the Plasmodium falciparum genome codes for genes with unknown functions, which presents a major challenge to understanding the parasite's biology. Large-scale functional analysis of the parasite genome is essential to pave the way for novel therapeutic intervention strategies against the disease and yet difficulties in genetic manipulation of this deadly human malaria parasite have been a major hindrance for functional analysis of its genome. Here, we used a forward functional genomic approach to study P. falciparum and identify genes important for optimal parasite development in the disease-causing, intraerythrocytic stages. We analyzed 123 piggyBac insertion mutants of P. falciparum for proliferation efficiency in the intraerythrocytic stages, in vitro. Almost 50% of the analyzed mutants showed significant reduction in proliferation efficiency, with 20% displaying severe defects. Functional categorization of genes in the severely attenuated mutants revealed significant enrichment for RNA binding proteins, suggesting the significance of post-transcriptional gene regulation in parasite development and emphasizing its importance as an antimalarial target. This study demonstrates the feasibility of much needed forward genetics approaches for P. falciparum to better characterize its genome and accelerate drug and vaccine development.

Nucleosome occupancy at transcription start sites in the human malaria parasite: A hard-wired evolution of virulence?

Almost a decade after the publication of the complete sequence of the genome of the human malaria parasite Plasmodium falciparum, the mechanisms involved in gene regulation remain poorly understood. Like other eukaryotic organisms, P. falciparum's genomic DNA organizes into nucleosomes. Nucleosomes are the basic structural units of eukaryotic chromatin and their regulation is known to play a key role in regulation of gene expression. Despite its importance, the relationship between nucleosome positioning and gene regulation in the malaria parasite has only been investigated recently. Using two independent and complementary techniques followed by next-generation high-throughput sequencing, our laboratory recently generated a dynamic atlas of nucleosome-bound and nucleosome-free regions (NFRs) at single-nucleotide resolution throughout the parasite erythrocytic cycle. We have found evidences that genome-wide changes in nucleosome occupancy play a critical role in controlling the rigorous parasite replication in infected red blood cells. However, the role of nucleosome positioning at remarkable locations such as transcriptional start sites (TSS) was not investigated. Here we show that a study of NFR in experimentally determined TSS and in silico-predicted promoters can provide deeper insights of how a transcriptionally permissive organization of chromatin can control the parasite's progression through its life cycle. We find that NFRs found at TSS and core promoters are strongly associated with high levels of gene expression in asexual erythrocytic stages, whereas nucleosome-bound TSSs and promoters are associated with silent genes preferentially expressed in sexual stages. The implications in terms of regulatory evolution, adaptation of gene expression and their impact in the design of antimalarial strategies are discussed.

Recent Progress in Functional Genomic Research in Plasmodium falciparum

With the completion and near completion of many malaria parasite genome-sequencing projects, efforts are now being directed to a better understanding of gene functions and to the discovery of vaccine and drug targets. Inter- and intraspecies comparisons of the parasite genomes will provide invaluable insights into parasite evolution, virulence, drug resistance, and immune invasion. Genome-wide searches for loci under various selection pressures may lead to discovery of genes conferring drug resistance or encoding for protective antigens. In addition, the Plasmodium falciparum genome sequence provides the basis for the development of various microarrays to monitor gene expression and to detect nucleotide substitution and deletion/amplification. Genome-wide profiling of the parasite proteome, chromatin modification, and nucleosome position also depend on availability of the parasite genome. In this brief review, we will highlight some recent advances and studies in characterizing gene function and related phenotype in P. falciparum that were made possible by the genome sequence, particularly the development of a genome-wide diversity map and various high-throughput genotyping methods for genome-wide association studies (GWAS).

Plasmodium Dihydroorotate Dehydrogenase: A Promising Target for Novel Anti-Malarial Chemotherapy

Malaria remains a globally prevalent infectious disease that leads to significant morbidity and mortality. While there are a number of drugs approved for its treatment, drug resistance has compromised most of them, making the development of new drugs for the treatment and prevention of malaria essential. The completion of the Plasmodium falciparum genome and a growing understanding of parasite biology are fueling the search for novel drug targets. Despite this, few targets have been chemically validated in vivo. The pyrimidine biosynthetic pathway illustrates one of the best examples of successful identification of anti-malarial drug targets. This review focuses on recent studies to exploit the fourth enzyme in the de novo pyrimidine biosynthetic pathway of P. falciparum, dihydroorotate dehydrogenase (PfDHODH), as a new target for drug discovery. Several chemical scaffolds have been identified by high throughput screening as potent inhibitors of PfDHODH and these show strong selectivity for the malarial enzyme over that from the human host. Potent activity against parasites in whole cell models with good correlation between activity on the enzyme and the parasite have also been observed for a number of the identified series. Lead optimization of a triazolopyrimidine-based series has identified an analog with prolonged plasma exposure, that is orally bioavailable, and which shows good efficacy against the in vivo mouse model of the disease. These data provide strong evidence that PfDHODH is a validated target for the identification of new antimalarial chemotherapy. The challenge remains to identify compounds with the necessary combination of potency and metabolic stability to allow identification of a clinical candidate.

A novel DEAD box helicase Has1p from Plasmodium falciparum: N-terminal is essential for activity

Helicases catalyze the opening of nucleic acid duplexes and are implicated in many nucleic acid metabolic cellular processes that require single stranded DNA or reorganization of RNA structure. Previously we have reported that Plasmodium falciparum genome contains a number of DEAD box helicases. In the present study we report the cloning, expression and characterization of one of the novel members of DEAD box family from P. falciparum. Our results indicate that it is a homologue of Has1p from yeast and it contains DNA and RNA unwinding, nucleic acid-dependent ATPase and RNA binding activities. This enzyme can utilize all the nucleosidetriphosphates (NTPs) and deoxy nucleosidetriphosphates (dNTPs) for its unwinding activity. Using a truncated derivative of this protein we further report that the N-terminal region of the protein is essentially required for its activity. These studies suggest that besides the conserved helicase domain the highly variable N-terminal region also contributes in the activity of the protein.

Proteases of Plasmodium falciparum as Potential Drug Targets and Inhibitors Thereof

Malaria, caused by protozoa of the genus Plasmodium, remains one of the most dreadful infectious diseases worldwide killing more than 1 million people per year. The emergence of multidrug-resistant parasites highly demands a steadfast and continuous search not only for new targets but also for new anti-infectives addressing the known ones. As proteases in general have been proven to be excellent drug targets and the development of inhibitors has frequently resulted in approved drugs, this review will only focus on the proteases of Plasmodium falciparum as drug targets. The completion of the sequencing of the Plasmodium falciparum genome in 2002 lead to the discovery of nearly 100 putative proteases encoded therein. Within this review, only those proteases and inhibitors thereof will be discussed in more detail, in which their biological function has been determined undoubtedly or in those cases, in which the development of specific inhibitors has significantly contributed to the understanding of the underlying biological role of the respective protease thus validating the role as promising drug target.

In silico and biological survey of transcription-associated proteins implicated in the transcriptional machinery during the erythrocytic development of Plasmodium falciparum

BackgroundMalaria is the most important parasitic disease in the world with approximately two million people dying every year, mostly due to Plasmodium falciparum infection. During its complex life cycle in the Anopheles vector and human host, the parasite requires the coordinated and modulated expression of diverse sets of genes involved in epigenetic, transcriptional and post-transcriptional regulation. However, despite the availability of the complete sequence of the Plasmodium falciparum genome, we are still quite ignorant about Plasmodium mechanisms of transcriptional gene regulation. This is due to the poor prediction of nuclear proteins, cognate DNA motifs and structures involved in transcription.ResultsA comprehensive directory of proteins reported to be potentially involved in Plasmodium transcriptional machinery was built from all in silico reports and databanks. The transcription-associated proteins were clustered in three main sets of factors: general transcription factors, chromatin-related proteins (structuring, remodelling and histone modifying enzymes), and specific transcription factors. Only a few of these factors have been molecularly analysed. Furthermore, from transcriptome and proteome data we modelled expression patterns of transcripts and corresponding proteins during the intra-erythrocytic cycle. Finally, an interactome of these proteins based either on in silico or on 2-yeast-hybrid experimental approaches is discussed.ConclusionThis is the first attempt to build a comprehensive directory of potential transcription-associated proteins in Plasmodium. In addition, all complete transcriptome, proteome and interactome raw data were re-analysed, compared and discussed for a better comprehension of the complex biological processes of Plasmodium falciparum transcriptional regulation during the erythrocytic development.

A genomic glimpse of aminoacyl-tRNA synthetases in malaria parasite Plasmodium falciparum

BackgroundPlasmodium parasites are causative agents of malaria which affects >500 million people and claims ~2 million lives annually. The completion of Plasmodium genome sequencing and availability of PlasmoDB database has provided a platform for systematic study of parasite genome. Aminoacyl-tRNA synthetases (aaRSs) are pivotal enzymes for protein translation and other vital cellular processes. We report an extensive analysis of the Plasmodium falciparum genome to identify and classify aaRSs in this organism.ResultsUsing various computational and bioinformatics tools, we have identified 37 aaRSs in P. falciparum. Our key observations are: (i) fraction of proteome dedicated to aaRSs in P. falciparum is very high compared to many other organisms; (ii) 23 out of 37 Pf-aaRS sequences contain signal peptides possibly directing them to different cellular organelles; (iii) expression profiles of Pf-aaRSs vary considerably at various life cycle stages of the parasite; (iv) several PfaaRSs posses very unusual domain architectures; (v) phylogenetic analyses reveal evolutionary relatedness of several parasite aaRSs to bacterial and plants aaRSs; (vi) three dimensional structural modelling has provided insights which could be exploited in inhibitor discovery against parasite aaRSs.ConclusionWe have identified 37 Pf-aaRSs based on our bioinformatics analysis. Our data reveal several unique attributes in this protein family. We have annotated all 37 Pf-aaRSs based on predicted localization, phylogenetics, domain architectures and their overall protein expression profiles. The sets of distinct features elaborated in this work will provide a platform for experimental dissection of this family of enzymes, possibly for the discovery of novel drugs against malaria.

Prediction of potential antimalarial targets of artemisinin based on protein information from whole genome of Plasmodium falciparum

On the basis of the genomic data and protein pathway information about Plasmodium falciparum clone 3D7 from the NCBI taxonomy database and the KEGG database, eight key protein enzymes in the signal pathways were selected to perform molecular docking with artemisinin. The binding modes obtained from the molecular docking suggested that purine nucleoside phosphorylase (pfPNP), peptide deformylase (pfPDF), and ribose 5-phosphate isomerase (pfRpiA) may be involved in the antimalarial mode of action of artemisinin. Artemisinin exhibited its antimalarial activity probably by interfering with the metabolic pathways of purine, pyrimidine, methionine, glyoxylate and dicarboxylate, or pentose phosphate.

Plasmodium falciparum: new molecular targets with potential for antimalarial drug development

Malaria remains one of the world’s most devastating infectious diseases. Drug resistance to all classes of antimalarial agents has now been observed, highlighting the need for new agents that act against novel parasite targets. The complete sequencing of the Plasmodium falciparum genome has allowed the identification of new molecular targets within the parasite that may be amenable to chemotherapeutic intervention. In this review, we investigate four possible targets for the future development of new classes of antimalarial agents. These targets include histone deacetylase, the aspartic proteases or plasmepsins, aminopeptidases and the purine salvage enzyme hypoxanthine–xanthine–guanine phosphoribosyltransferase.

Malaria Vaccines: Where Next?

A malaria vaccine for mass immuniza-tion, which could be delivered cheaply andwidely and provide long lasting protection,would have a massive effect on globalpublic health. However, such a vaccinedoes not currently exist. The reduction inglobal malaria incidence, in part due tothe ongoing introduction of mosquitocontrol measures such as insecticide-treat-ed nets and disease treatment with arte-mesinin combination drug therapy, raisesthe performance requirements for a futurevaccine, since the cost may be high relativeto that of other interventions and the scaleof the disease burden. Nevertheless, histo-ry teaches that complacency and an over-reliance on a small number of tools tocombat this disease are dangerous. Thereis therefore still a pressing need for avaccine to complement other control andpotential elimination tools. The big ques-tion is whether or not a malaria vaccinethat fulfills the above criteria can bedeveloped.RTS,S, the first malaria vaccine thattargets the so-called pre-erythrocytic stagesin humans, has just started in Phase 3 trials[1]; this is a major achievement based onnumerous trials (most recent trials in [2,3])and 40 years of research (reviewed in [4]).Although questions persist about whetherthis vaccine will be effective enough [5]and about the nature of the protectivemechanisms, does this achievement signalthe end for research on new vaccines andthe basic parasite biology and host immu-nology that underpins vaccine research?Other malaria vaccine candidates thattarget the asexual blood stages are not soadvanced, and without improvement maynot live up to hopes and expectations[6,7]; is it therefore time to reassess thestrategy on which they were developed[8]? For example, instead of subunit orvectored vaccines, the potential use of liveor live and attenuated parasite vaccineshas been proposed [9,10] and is beingpursued (http://www.sanaria.com/). Nowthat the international community is striv-ing to eliminate malaria, maybe therequirements for a malaria vaccine arechanging. Perhaps now is the time for agreater effort to research and develop thenext generation of malaria vaccines. If thisis the time for reinvigoration, what moredo we need to know to develop malariavaccines?Current vaccines are based on a handfulof proteins, several of which were firstdescribed several decades ago and beforeanalysis of the Plasmodium falciparum ge-nome indicated that there are about 5,400protein-coding genes, some of which areexpressed in an exquisitely stage-specificmanner and others that are not. Are anyof these other proteins antigens that areworth considering for vaccine develop-ment? It is unlikely that the level ofresources devoted to the circumsporozoitesurface protein (CSP) development thatled to RTS,S could be mustered to supportthe further development of any newantigen. If all new malaria vaccines needto be compared against RTS,S, this couldonly be done in an expensive clinical trialformat since alternative assays of efficacydo not exist. The cost and practicalities ofthis may inhibit vaccine-related researchand the development of next generationvaccines, because a company or public–private partnership may not be preparedto put in the resources to produce a newvaccine. We must, therefore, ask wherelimited resources are best placed; vaccinediscovery and development are bothexpensive. We have to take rationalapproaches; it’s the best we can do, andresources are not available for purelyempirical approaches. We need to en-hance efforts on basic science in combi-nation with clinical studies to provide astrong rationale for further vaccine devel-opment. This will need to be an integratedrather than a compartmentalized ap-proach; vaccine development from antigendiscovery to clinical trial is not a linearprocess. For example, clinical samplescollected as part of a vaccine evaluationtrial are essential for the development ofbetter and more appropriate methods andassays to understand relevant humanimmune responses.

Isolation and functional characterization of eIF4F components and poly(A)-binding protein from Plasmodium falciparum

The multisubunit translation initiation complex eIF4F contains eIF4E, eIF4A and eIF4G. eIF4A is an ATP-dependent RNA helicase. eIF4G provides the platform for binding initiation factors and it contains the binding sites for eIF4A and eIF4E and interacts with poly(A)-binding protein (PABP). Although the genome of Plasmodium falciparum is fully sequenced but the gene annotation is still incomplete. In this manuscript we present the isolation and characterization of components of the eIF4F complex i.e. eIF4E, eIF4G and PABP from P. falciparum. Our studies indicate that PfeIF4E is involved in translation and PfPABP binds poly(A) specifically. We demonstrate the interaction of PfeIF4G with PfeIF4E, PfeIF4A (PfH45) and PfPABP. These studies demonstrate that these factors are structurally and functionally conserved. These studies will contribute to understand the important process and components of translation complex in the malaria parasite.

Clonally variant gene families in Plasmodium falciparum share a common activation factor

SummaryThe genome of the malaria parasite Plasmodium falciparum contains several multicopy gene families, including var, rifin, stevor and Pfmc‐2TM. These gene families undergo expression switching and appear to play a role in antigenic variation. It has recently been shown that forcing parasites to express high copy numbers of transcriptionally active, episomal var promoters led to gradual downregulation and eventual silencing of the entire var gene family, suggesting that a limiting titratable factor plays a role in var gene activation. Through similar experiments using rifin, stevor or Pfmc‐2TM episomal promoters we show that promoter titration can be used as a general method to downregulate multicopy gene families in P. falciparum. Additionally, we show that promoter titration with var, rifin, stevor or Pfmc‐2TM episomal promoters results in downregulation of expression not only of the family to which the episomal promoter belongs, but also members of the other gene families, suggesting that the var‐specific titratable factor previously described is shared by all four families. Further, transcriptionally active promoters from different families colocalize within the same subnuclear expression site, indicating that the role that nuclear architecture plays in var gene regulation also likely applies to the other multicopy gene families of P. falciparum.

Localization of the ATP-binding cassette (ABC) transport proteins PfMRP1, PfMRP2, and PfMDR5 at the Plasmodium falciparum plasma membrane

BackgroundThe spread of drug resistance has been a major obstacle to the control of malaria. The mechanisms underlying drug resistance in malaria seem to be complex and multigenic. The current literature on multiple drug resistance against anti-malarials has documented PfMDR1, an ATP-binding cassette (ABC) protein, as an important determinant of resistance. In the Plasmodium falciparum genome, there are several ABC transporters some of which could be putative drug transporting proteins. In order to understand the molecular mechanisms underlying drug resistance, characterization of these transporters is essential. The aim of this study was to characterize and localize putative ABC transporters.MethodsIn the plasmoDB database, 16 members of the P. falciparum ABC family can be identified, 11 of which are putative transport proteins. A phylogenetic analysis of the aligned NBDs of the PfABC genes was performed. Antibodies against PfMRP1 (PfABCC1), PfMRP2 (PfABCC2), and PfMDR5 (PfABCB5) were generated, affinity purified and used in immunocytochemistry to localize the proteins in the asexual stages of the parasite.ResultsThe ABC family members of P. falciparum were categorized into subfamilies. The ABC B subfamily was the largest and contained seven members. Other family members that could be involved in drug transport are PfABCC1, PfABCC2, PfABCG1, and PfABCI3. The expression and localization of three ABC transport proteins was determined. PfMRP1, PfMRP2, and PfMDR5 are localized to the plasma membrane in all asexual stages of the parasite.ConclusionIn conclusion, 11 of the 16 ABC proteins in the P. falciparum genome are putative transport proteins, some of which might be involved in drug resistance. Moreover, it was demonstrated that three of these proteins are expressed on the parasite's plasma membrane.

Identification of “Missing” Metabolic Proteins of Plasmodium falciparum: A Bioinformatics Approach (SUPPLEMENTARY INFORMATION)

The genome of Plasmodium falciparum, a malarial parasite, is atypical as many of the encoded proteins have diverged extensively from their homologues in other organisms. Hence homology-based information transfer is not entirely successful and presently, proper function annotation is unavailable for over 50% of the proteome. It has been hypothesized that enzymes participating in nearly 69 metabolic steps are not yet identified. In this paper we report detection of some of the "missing metabolic enzymes" of P. falciparum. Our approach for remote homology detection to recognize the "missing" P. falciparum enzymes employs multiple profiles for every protein domain family. A blind assessment of the approach to recognize known metabolic proteins of P. falciparum resulted in 94% success rate. Using this approach we have successfully recognized 14 of the "missing" enzymes. Results of protein fold recognition and information from microarray and protein-protein interaction datasets are consistent with our predictions. In a few cases we also provide the list of functionally important residues extrapolated on the basis of homology.