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Information on EC 4.99.1.8 - heme ligase
for references in articles please use BRENDA:EC4.99.1.8
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EC Tree 4 Lyases
4.99 Other lyases
4.99.1 Sole sub-subclass for lyases that do not belong in the other subclasses
4.99.1.8 heme ligase
IUBMB Comments
This heme detoxifying enzyme is found in Plasmodium parasites and converts toxic heme to crystalline hemozoin. These organisms lack the mammalian heme oxygenase for elimination of heme.
The expected taxonomic range for this enzyme is: Plasmodium
- 4.99.1.8
- plasmodium
- malaria
- hemozoin
- falciparum
- hemoglobin
-
antimalarial
- vacuole
- chloroquine
-
detoxify
- medicine
- artemisinins
-
reductase-thymidylate
-
byproduct
- dihydrofolate
-
heme-binding
- polymerization
- yoelii
- analysis
showSynonyms
heme detoxification protein, heme-detoxification protein, heme ligase, more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
HDP
heme detoxification protein
heme detoxification protein
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 ferriprotoporphyrin IX = beta-hematin
-
-
-
-
2 ferriprotoporphyrin IX = beta-hematin
HDP-mediated transformation of heme into hemozoin is not a single-step process, and involves a transient intermediate, most likely a cyclic heme dimer
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PATHWAY SOURCE
PATHWAYS
MetaCyc
hemoglobin degradation
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SYSTEMATIC NAME
IUBMB Comments
Fe3+:ferriprotoporphyrin IX ligase (beta-hematin-forming)
This heme detoxifying enzyme is found in Plasmodium parasites and converts toxic heme to crystalline hemozoin. These organisms lack the mammalian heme oxygenase for elimination of heme.
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2 ferriprotoporphyrin IX
beta-hematin
Substrates: -
Products: -
Products: -
?
additional information
?
-
Substrates: the kinetics and the manner of HDP-mediated hemozoin production are dependent on the substrate concentration, and a small fraction of substrate remains untransformed to hemozoin irrespective of reaction time. Optimization of the pyridine hemochrome assay to measure the HDP-mediated transformation of free heme into hemozoin (Hz). The assay is performed at pH 5.2, considering the physiological environment inside the Plasmodium acidic food vacuole (pH 5.2-5.4), the site for hemoglobin digestion. Heme forms a sticky aggregate under such acidic environment. Introduction of 5% v/v DMSO in the reaction mixture facilitates a more homogeneous distribution of heme suspension
Products: -
Products: -
-
ferriprotoporphyrin IX
beta-hematin
-
Substrates: hemozoin consists of an unusual polymer of hemes linked between the central ferric ion of one heme and a carboxylate side-group oxygen of another. The hemes are sequestered via this linkage into an insoluble product, providing a unique way for the malaria parasite to avoid the toxicity associated with soluble heme
Products: -
Products: -
?
ferriprotoporphyrin IX
beta-hematin
-
Substrates: hemozoin consists of an unusual polymer of hemes linked between the central ferric ion of one heme and a carboxylate side-group oxygen of another
Products: -
Products: -
?
ferriprotoporphyrin IX
beta-hematin
-
Substrates: -
Products: beta-hematin is also called hemozoin
Products: beta-hematin is also called hemozoin
?
ferriprotoporphyrin IX
beta-hematin
-
Substrates: -
Products: beta-hematin is also called hemozoin or Malaria pigment
Products: beta-hematin is also called hemozoin or Malaria pigment
?
ferriprotoporphyrin IX
beta-hematin
-
Substrates: HDP possesses 2.7 heme binding sites
Products: -
Products: -
?
heme
hemozoin
Substrates: most efficient substrate
Products: -
Products: -
?
heme
hemozoin
Substrates: enzyme binds heme with a very high affinity, KD value 89 nM
Products: -
Products: -
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2 ferriprotoporphyrin IX
beta-hematin
Substrates: -
Products: -
Products: -
?
ferriprotoporphyrin IX
beta-hematin
-
Substrates: hemozoin consists of an unusual polymer of hemes linked between the central ferric ion of one heme and a carboxylate side-group oxygen of another. The hemes are sequestered via this linkage into an insoluble product, providing a unique way for the malaria parasite to avoid the toxicity associated with soluble heme
Products: -
Products: -
?
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
diethyldicarbonate
histidine modification using diethyldicarbonate leads to reduction in heme binding and hemozoin formation
N-[2-[(7-methyl-2,3-dihydro-1H-inden-4-yl)oxy]-3-pyridinyl]-3,4-dihydro-2H-1,5-benzodioxepine-7-carboxamide
compound binds the modeled HDP structure in the heme/hemoglobin-binding pockets, and inhibits parasite growth in a dose-dependent manner
additional information
not inhibitory up to 0.04 mM: pyrimethamine
-
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DISEASE
TITLE OF PUBLICATION
LINK TO PUBMED
Infections
Evaluation of antigen detection tests, microscopy, and polymerase chain reaction for diagnosis of malaria in peripheral blood in asymptomatic pregnant women in nanoro, burkina faso.
Malaria
Cloning, expression and functional characterization of heme detoxification protein (HDP) from the rodent malaria parasite Plasmodium vinckei.
Malaria
Evaluation of antigen detection tests, microscopy, and polymerase chain reaction for diagnosis of malaria in peripheral blood in asymptomatic pregnant women in nanoro, burkina faso.
Malaria
New developments in malaria diagnostics: Monoclonal antibodies against Plasmodium dihydrofolate reductase-thymidylate synthase, heme detoxification protein and glutamate rich protein.
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5.2
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
3.3 - 5.2
-
pH 3.3-4.4: optimum, pH 5.2: about 75% of maximal activity, no activity above pH 5.6
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
expression is increased in late trophozoite and schizont stages of parasite
brenda
additional information
enzyme is expressed throughout the intra erythrocytic stages
brenda
expression is increased in late trophozoite and schizont stages of parasite
brenda
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
the parasite utilizes a circuitous outbound-inbound trafficking route by initially secreting HDP into the cytosol of infected red blood cells
-
brenda
additional information
indirect immunofluorescence assay of transgenic PfHDP-GFP asexual blood stages shows that PfHDP is localized in the punctate structures trafficked to the food vacuole. The enzyme is also localized in infected erythrocyte cytosol for its role in hemoglobin (Hb) uptake, association of PfHDP with Hb as well as with the members of PTEX translocon complex, e.g. Pfexp-2. PfHDP lacks a classical N-terminal signal sequence or PEXEL motif, which usually assists in the sorting and transporting of any protein to a destined site using the translocon complex. The translocon complex consisting of PTEX150, exportin 2, PTEX88, HSP101, and Trx2, is used by the parasite to export proteins from the parasite to the erythrocyte cytoplasm
-
brenda
falcipain 2 coexists with heme detoxification protein and forms a hemozoin formation complex in the Plasmodium food vacuole
brenda
Highest Expressing Human Cell Lines
Cell Line LinksPlease wait a moment until the data is sorted. This message will disappear when the data is sorted.
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
malfunction
physiological function
malfunction
among all known mediators of Hz production, the heme detoxification protein (HDP) is considered the most efficient in Hz production. The enzyme is strictly conserved in all Plasmodium species, and a parasite with gene knockout for this protein is not viable in Plasmodium falciparum
malfunction
PfHDP knockdown significantly reduces the hemoglobin (Hb) uptake in PfHDP-HA-GlmS transgenic parasites in comparison to the wild-type parasites. Morphological analysis of PfHDP-HA-GlmS transgenic parasites in the presence of GlcN show food vacuole abnormalities and parasite stress, thereby causing a growth defect in the development of these parasites. Transient knockdown of a member of translocon complex, PfHSP101 in HSP101-DDDHA parasites also show a decreased uptake of Hb inside the parasite. Parasites of the parasitePfHSP101-DDDHA knockdown line expressing low levels of PfHSP101 protein display low levels of Hb inside the parasite. HSP101 DDDHA knockdown parasites take up less Hb from the host erythrocyte cytoplasm
physiological function
-
hemozoin is the main protective mechanism against heme toxicity in malaria parasites
physiological function
identification of two heme binding sites and a hemoglobin binding site in Pf HDP. Treatment of Plasmodium falciparum 3D7 parasites with peptide corresponding to the hemoglobin binding domain results in food vacuole abnormalities similar to that seen with cysteine protease inhibitor, E-64
physiological function
non-crystalline heme is stored within hemoglobin in the digestive vacuole, and then converted to crystallized heme with the assistance of the heme detoxification protein. A coupling exists between the rate of hemoglobin digestion and the rate of heme dimerization and crystallization. In vivo heme crystallization occurs at a rate of about 1000/s, which is close to the in vitro rate of heme dimerization measured for the heme detoxification protein (HDP). HDP may be primarily responsible for heme crystallization
physiological function
most antimalarial therapeutics, including chloroquine and artemisinin, induce free heme-mediated toxicity in Plasmodium. This cytotoxic heme is produced as a by-product during the large-scale digestion of host hemoglobin. Conversion of this host-derived heme into inert crystalline hemozoin is the only defense mechanism in Plasmodium against heme-induced cytotoxicity. parasite degrades around 75% of the hemoglobin in an infected erythrocyte. The massive hemoglobin digestion in the parasite's food vacuole liberates copious amounts of free heme prosthetic group. As a result, most host-derived heme remains within the food vacuole and accumulates. This free heme, especially at such a high concentration, can impose cytotoxicity based on inducing oxidant-mediated cellular damage. Heme detoxification protein (HDP), a highly conserved plasmodial protein, is reported to be the most efficient biological mediator for heme to hemozoin transformation
physiological function
hemoglobin (Hb) degradation is crucial for the growth and survival of Plasmodium falciparum in human erythrocytes. During the asexual stage of life cycle inside the human erythrocyte, the parasite digests Hb in a specialized organelle, the food vacuole. Hb is digested by the sequential action of a complex of proteases including plasmepsins, falcipains, and falcilysin inside the food vacuole. These proteases cleave Hb to short peptides and finally to amino acids. The free toxic by-product, heme generated during the process is subsequently converted into an inert insoluble polymer, hemozoin, by the enzyme HDP. Role of heme detoxification protein (PfHDP) in Hb uptake and parasite development, interaction between PfHDP and the translocon complex at the parasitophorus vacuole membrane. HDP is extremely potent in converting heme to hemozoin. PfHDP, a food vacuole-associated protein, possesses two heme-binding sites and a Hb-binding site and is a part of the 200 kDa complex with other proteins including falcipain 2/2, plasmepsin II, plasmepsin IV, and histo-aspartic protease inside the food vacuole
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UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). HDP_PLAF7
205
0
24337
Swiss-Prot
other Location (Reliability: 1)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
x * 50000, about, recombinant GFP-tagged HDP, SDS-PAGE, x * 24300, HDP, SDS-PAGE
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
H192A
the mutant shows decreased hemozoin formation activity compared to the wild type enzyme
H44A
the mutant shows decreased hemozoin formation activity compared to the wild type enzyme
H44A/H58A/H70A/H79A/H122A/H172A/H175A/H192A/H197A
the mutant shows less than half of the wild type activity
H58A
the mutant shows decreased hemozoin formation activity compared to the wild type enzyme
H70A
the mutant shows decreased hemozoin formation activity compared to the wild type enzyme
H79A
the mutant shows decreased hemozoin formation activity compared to the wild type enzyme
additional information
additional information
-
the truncated enzyme proteins HDP3 (encoded by amino acids 88-205 of the full-length protein, representing the fasciclin domain) and HDP2 (encoded by amino acids 187 and lacking the fasciclin domain) are unable to produce hemozoin. The full-length enzyme is required for heme binding and hemozoin production activities of the protein
additional information
knocking down the HDP mRNA with glucosamine, resulting in PfHDP-HA-GlmS transgenic. The effect of knockdown on parasite invasion is evaluated with 3D7 strain of Plasmodium falciparum as the control
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
expression in Escherichia coli
expression of full length enzyme in Escherichia coli, expression of two truncated enzyme proteins (HDP3 encoded by amino acids 88-205 of the full-length protein, representing the fasciclin domain and HDP2 encoded by amino acids 187 and lacking the fasciclin domain)
-
recombinant overexpression of C-terminally GFP-tagged enzyme is expressed in an episomal construct from the pSSPF2 vector with GFP tag at 3'end under the control of HSP86 promoter in transgenic Plasmodium falciparum blood-stage parasites
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RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
analysis
development of a high resolution correlative combination of cryo soft X-ray tomography to obtain 3D parasite ultrastructure with cryo X-ray fluorescence microscopy to measure heme concentrations
drug development
the enzyme is a target for drug development. Combining HDP as a catalyst and the pyridine hemochrome assay will facilitate the efficient screening of future antimalarials
medicine
medicine
-
involvement of heme detoxification protein in the process of formation of hemozoin suggests that it could be a malaria drug target
medicine
-
involvement of heme detoxification protein in the process of formation of hemozoin suggests that it could be a malaria drug target
medicine
-
HDP is a conserved target for future antimalarial development
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Vinayak, S.; Rathore, D.; Kariuki, S.; Slutsker, L.; Shi, Y.P.; Villegas, L.; Escalante, A.A.; Udhayakumar, V.
Limited genetic variation in the Plasmodium falciparum heme detoxification protein (HDP)
Infect. Genet. Evol.
9
286-289
2009
Plasmodium falciparum
brenda
Jani, D.; Nagakatti, R.; Beatty, W.; Angel, R.; Slebodnick, C.; Andersen, J.; Kumar, S.; Rathore, D.
HDP - a novel heme detoxification protein from the malaria parasite
PLos Pathog.
4
e1000053
2008
Plasmodium falciparum, Plasmodium yoelii
brenda
Slater, A.F.G.; Swiggard, W.J.; Orton, B.R.; Flitter, W.D.; Goldberg, D.E.; Cerami, A.; Henderson, G.B.
An iron-carboxylate bond links the heme units of malaria pigment
Proc. Natl. Acad. Sci. USA
88
325-329
1991
Plasmodium falciparum
brenda
Wagner, H.
Hemozoin: malaria's built-in adjuvant and TLR9 agonist
Cell Host Microbe
7
5-6
2010
Plasmodium falciparum
brenda
Shio, M.; Kassa, F.; Bellemare, M.; Olivier, M.
Innate inflammatory response to the malarial pigment hemozoin
Microbes Infect.
12
889-899
2010
Plasmodium falciparum
brenda
Nakatani, K.; Ishikawa, H.; Aono, S.; Mizutani, Y.
Heme-binding properties of heme detoxification protein from Plasmodium falciparum
Biochem. Biophys. Res. Commun.
439
477-480
2013
Plasmodium falciparum (Q8IL04), Plasmodium falciparum
brenda
Kattenberg, J.H.; Versteeg, I.; Migchelsen, S.J.; Gonzalez, I.J.; Perkins, M.D.; Mens, P.F.; Schallig, H.D.
New developments in malaria diagnostics: monoclonal antibodies against Plasmodium dihydrofolate reductase-thymidylate synthase, heme detoxification protein and glutamate rich protein
mAbs
4
120-126
2012
Plasmodium falciparum (Q8IL04), Plasmodium falciparum, Plasmodium falciparum NF54 (Q8IL04)
brenda
Chugh, M.; Sundararaman, V.; Kumar, S.; Reddy, V.S.; Siddiqui, W.A.; Stuart, K.D.; Malhotra, P.
Protein complex directs hemoglobin-to-hemozoin formation in Plasmodium falciparum
Proc. Natl. Acad. Sci. USA
110
5392-5397
2013
Plasmodium falciparum (Q8IL04), Plasmodium falciparum
brenda
Nakatani, K.; Ishikawa, H.; Aono, S.; Mizutani, Y.
Identification of essential histidine residues involved in heme binding and hemozoin formation in heme detoxification protein from Plasmodium falciparum
Sci. Rep.
4
6137
2014
Plasmodium falciparum (Q8IL04), Plasmodium falciparum
brenda
Soni, A.; Goyal, M.; Prakash, K.; Bhardwaj, J.; Siddiqui, A.J.; Puri, S.K.
Cloning, expression and functional characterization of heme detoxification protein (HDP) from the rodent malaria parasite Plasmodium vinckei
Gene
566
109-119
2015
Plasmodium vinckei petteri (F5BWX4)
brenda
Gupta, P.; Mehrotra, S.; Sharma, A.; Chugh, M.; Pandey, R.; Kaushik, A.; Khurana, S.; Srivastava, N.; Srivastava, T.; Deshmukh, A.; Panda, A.; Aggarwal, P.; Bhavesh, N.S.; Bhatnagar, R.K.; Mohmmed, A.; Gupta, D.; Malhotra, P.
Exploring heme and hemoglobin binding regions of Plasmodium heme detoxification protein for new antimalarial discovery
J. Med. Chem.
60
8298-8308
2017
Plasmodium falciparum (Q8IL04)
brenda
Kapishnikov, S.; Grolimund, D.; Schneider, G.; Pereiro, E.; McNally, J.; Als-Nielsen, J.; Leiserowitz, L.
Unraveling heme detoxification in the malaria parasite by in situ correlative X-ray fluorescence microscopy and soft X-ray tomography
Sci. Rep.
7
7610
2017
Plasmodium falciparum (Q8IL04), Plasmodium falciparum
brenda
Singh, R.; Makde, R.D.
An assay procedure to investigate the transformation of toxic heme into inert hemozoin via plasmodial heme detoxification protein
Biochim. Biophys. Acta
1870
140832
2022
Plasmodium falciparum (Q8IL04)
brenda
Gupta, P.; Pandey, R.; Thakur, V.; Parveen, S.; Kaur, I.; Panda, A.; Bishi, R.; Mehrotra, S.; Akhtar, A.; Gupta, D.; Mohmmed, A.; Malhotra, P.
Heme detoxification protein (PfHDP) is essential for the hemoglobin uptake and metabolism in Plasmodium falciparum
FASEB Bioadv.
4
662-674
2022
Plasmodium falciparum (Q8IL04)
brenda
EXTERNAL LINKS (specific for EC-Number 4.99.1.8)
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