Unique properties of Plasmodium falciparum porphobilinogen deaminase

The hybrid pathway for heme biosynthesis in the malarial parasite proposes the involvement of parasite genome-coded enzymes of the pathway localized in different compartments such as apicoplast, mitochondria, and cytosol. However, knowledge on the functionality and localization of many of these enzymes is not available. In this study, we demonstrate that porphobilinogen deaminase encoded by the Plasmodium falciparum genome (PfPBGD) has several unique biochemical properties. Studies carried out with PfPBGD partially purified from parasite membrane fraction, as well as recombinant PfPBGD lacking N-terminal 64 amino acids expressed and purified from Escherichia coli cells (ΔPfPBGD), indicate that both the proteins are catalytically active. Surprisingly, PfPBGD catalyzes the conversion of porphobilinogen to uroporphyrinogen III (UROGEN III), indicating that it also possesses uroporphyrinogen III synthase (UROS) activity, catalyzing the next step. This obviates the necessity to have a separate gene for UROS that has not been so far annotated in the parasite genome. Interestingly, ΔPfP-BGD gives rise to UROGEN III even after heat treatment, although UROS from other sources is known to be heat-sensitive. Based on the analysis of active site residues, a ΔPfPBGDL116K mutant enzyme was created and the specific activity of this recombinant mutant enzyme is 5-fold higher than ΔPfPBGD. More interestingly, ΔPfPBGDL116K catalyzes the formation of uroporphyrinogen I (UROGEN I) in addition to UROGEN III, indicating that with increased PBGD activity the UROS activity of PBGD may perhaps become rate-limiting, thus leading to non-enzymatic cyclization of preuroporphyrinogen to UROGEN I. PfPBGD is localized to the apicoplast and is catalytically very inefficient compared with the host red cell enzyme

Localization of Heme Biosynthesis Pathway Enzymes in Plasmodium falciparum

365-373Protein trafficking in the malarial parasite Plasmodium falciparum is dictated by a complex life-cycle that involves a variety of intra-cellular and host cell destinations, such as the mitochondrion, apicoplast, rhoptries and micronemes. Of these, the apicoplast and mitochondrion are believed to account for more than 10% of this traffic. Studies have shown that mechanisms for mitochondrion and apicoplast targeting are distinct, despite their close physical proximity. The heme biosynthesis pathway spans both these organelles, making trafficking studies crucial for the spatial demarcation of the constituent interactions. This minireview highlights the challenges in identifying the possible sub-cellular destinations of the heme pathway enzymes using gleanings from literature survey as well as focussed bioinformatic analysis

IPSVAC – Integrated Platform for Sequence Visualization, Analysis and Comparison

Motivation: With the increased availability of sequence information and concomitant increase in the number of automated analysis servers, biologists today need to deal with multiple data sources and multiple software tools which use diverse methods and algorithms for analysis. For knowledge mining and hypothesis building exercises, user level intervention in terms of comparing, validating and visualizing second order features derived from the sequence data is of crucial importance. Convenient options for doing this from an integrated platform which enables the user to operate with a single input format and to retrieve parsed outputs, relevant to his research context, from different servers in customizable user defined visual formats for easy comparison and analysis are an urgent requirement for biologists in the post genomic era. The seemingly inevitable necessity of having to negotiate with heterogeneous legacy resources, which have come up because of rapid parallel developments on all fronts related to technology, approach and algorithms, constitutes the essential challenge involved. Results: IPSVAC is an integrated toolkit, which is interactive, customizable and modular. It can be used for analysis; display and storage of results, related to biological sequence data, obtained from a choice of tools which may be available publicly or which have been incorporated as an add-on module

Unique properties of plasmodium falciparum porphobilinogen deaminase

The hybrid pathway for heme biosynthesis in the malarial parasite proposes the involvement of parasite genome-coded enzymes of the pathway localized in different compartments such as apicoplast, mitochondria, and cytosol. However, knowledge on the functionality and localization of many of these enzymes is not available. In this study, we demonstrate that porphobilinogen deaminase encoded by the Plasmodium falciparum genome (PfPBGD) has several unique biochemical properties. Studies carried out with PfPBGD partially purified from parasite membrane fraction, as well as recombinant PfPBGD lacking N-terminal 64 amino acids expressed and purified from Escherichia coli cells ( \Delta PfPBGD), indicate that both the proteins are catalytically active. Surprisingly, PfPBGD catalyzes the conversion of porphobilinogen to uroporphyrinogen III (UROGEN III), indicating that it also possesses uroporphyrinogen III synthase (UROS) activity, catalyzing the next step. This obviates the necessity to have a separate gene for UROS that has not been so far annotated in the parasite genome. Interestingly, \Delta PfP-BGD gives rise to UROGEN III even after heat treatment, although UROS from other sources is known to be heat-sensitive. Based on the analysis of active site residues, a \Delta PfPBGDL116K mutant enzyme was created and the specific activity of this recombinant mutant enzyme is 5-fold higher than \Delta PfPBGD. More interestingly, \Delta PfPBGDL116K catalyzes the formation of uroporphyrinogen I (UROGEN I) in addition to UROGEN III, indicating that with increased PBGD activity the UROS activity of PBGD may perhaps become rate-limiting, thus leading to non-enzymatic cyclization of preuroporphyrinogen to UROGEN I. PfPBGD is localized to the apicoplast and is catalytically very inefficient compared with the host red cell enzyme

CAGI, the Critical Assessment of Genome Interpretation, establishes progress and prospects for computational genetic variant interpretation methods

BackgroundThe Critical Assessment of Genome Interpretation (CAGI) aims to advance the state-of-the-art for computational prediction of genetic variant impact, particularly where relevant to disease. The five complete editions of the CAGI community experiment comprised 50 challenges, in which participants made blind predictions of phenotypes from genetic data, and these were evaluated by independent assessors.ResultsPerformance was particularly strong for clinical pathogenic variants, including some difficult-to-diagnose cases, and extends to interpretation of cancer-related variants. Missense variant interpretation methods were able to estimate biochemical effects with increasing accuracy. Assessment of methods for regulatory variants and complex trait disease risk was less definitive and indicates performance potentially suitable for auxiliary use in the clinic.ConclusionsResults show that while current methods are imperfect, they have major utility for research and clinical applications. Emerging methods and increasingly large, robust datasets for training and assessment promise further progress ahead