Purification and properties of uroporphyrinogen III synthase (co-synthase) from an overproducing recombinant strain of <i>Escherichia coli</i> K-12

Alwan, Asam F.; Mgbeje, B I A; Jordan, Peter M.

doi:10.1042/bj2640397

Cited by 49 publications

(18 citation statements)

References 23 publications

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“…The present study indicates that PfPBGD also has UROS activity leading to the formation of UROGEN III. While information on an independent UROS gene and purification of the enzyme is available from different sources such as E. coli (33), yeast (34), and the human (35) including its crystal structure (36), there have been no reports on the enzyme or gene from algal and plant sources (37). Interestingly, it has been reported that in Leptospira interrogans hemC codes for a bifunctional PBGD/UROS enzyme (38).…”

Section: Discussionmentioning

confidence: 99%

Unique Properties of Plasmodium falciparum Porphobilinogen Deaminase

Nagaraj

Arumugam

Bulusu

et al. 2008

Journal of Biological Chemistry

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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 (⌬PfPBGD), indicate that both the proteins are catalytically active. Surprisingly, PfPBGD catalyzes the conversion of porphobilinogen to uroporphyrinogen III (URO-GEN 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, ⌬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 ⌬PfPBGDL116K mutant enzyme was created and the specific activity of this recombinant mutant enzyme is 5-fold higher than ⌬PfPBGD. More interestingly, ⌬PfPBGDL116K catalyzes the formation of uroporphyrinogen I (UROGEN I) in addition to URO-GEN 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 URO-GEN I. PfPBGD is localized to the apicoplast and is catalytically very inefficient compared with the host red cell enzyme.Detailed studies of the metabolic pathways in the malarial parasite hold promise for the identification of new antimalarial drug targets (1, 2). Earlier studies in this laboratory had shown that the malarial parasite synthesizes heme de novo, despite acquiring heme from the host red cell hemoglobin in the intraerythrocytic stage. It has been shown that Plasmodium falciparum contains ␦-aminolevulinate dehydratase (ALAD) 2 of dual origin: one species encoded by the parasite genome (PfALAD) and another imported from the host red cell. Inhibition of ALAD activity in the parasite, the second enzyme of the pathway with a specific inhibitor, succinylacetone, leads to inhibition of heme synthesis and death of the parasite, indicating the potential of the pathway as a drug target (3, 4).Heme biosynthesis from glycine and succinyl-CoA involves eight different steps and the genes for all the enzymes of the pathway, with the exception of uroporphyrinogen III synthase (UROS, hemD) have been located on the parasite genome (5). However, a hemD orthologue has been identified in Toxoplasma gondii and is expected to be targeted to the apicoplast, a chloroplast-like organelle in the parasite (6). Based mostly on bioinformatics-based predictions, some e...

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Section: Discussionmentioning

confidence: 99%

Unique Properties of Plasmodium falciparum Porphobilinogen Deaminase

Nagaraj

Arumugam

Bulusu

et al. 2008

Journal of Biological Chemistry

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“…The advent of recombinant-DNA technology allowed the enzyme to be produced in much larger quantities (139,140), and the development of a fluorescencebased assay (141) coupled with more accurate high-performance liquid chromatography (HPLC) analysis to separate the type I and III isomers made the assays much more rapid and accurate (142). Consequently, after the initial laborious methods to isolate the first purified UroS enzyme from human blood (143), quite a few UroSs from a variety of organisms were subsequently purified as a consequence of recombinant-DNA approaches (144)(145)(146)(147). UroSs are generally monomeric species with molecular masses of around 30 kDa.…”

Section: Dailey Et Almentioning

confidence: 99%

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

Dailey

Gerdes³

et al. 2017

Microbiol Mol Biol Rev

273

335

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SUMMARY The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process. Bacteria utilize the initial core pathway but then add one additional common step to produce coproporphyrinogen III. Following this step, Gram-positive organisms oxidize coproporphyrinogen III to coproporphyrin III, insert iron to make coproheme, and finally decarboxylate coproheme to protoheme, whereas Gram-negative bacteria first decarboxylate coproporphyrinogen III to protoporphyrinogen IX and then oxidize this to protoporphyrin IX prior to metal insertion to make protoheme. In order to adapt to oxygen-deficient conditions, two steps in the bacterial pathways have multiple forms to accommodate oxidative reactions in an anaerobic environment. The regulation of these pathways reflects the diversity of bacterial metabolism. This diversity, along with the late recognition that three pathways exist, has significantly slowed advances in this field such that no single organism's heme synthesis pathway regulation is currently completely characterized.

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“…3 ( Fig. 2A) which is higher than that observed for the E. coli (pH 7.8) [8] and human (pH 7.4) [6] enzymes although similar to that obtained for the enzyme isolated from Euglena (pH 8.25) [5]. Due to the increased stability of uro'gen I11 synthase from B. subtilis, it was possible to investigate the optimum temperature for enzymic activity (Fig.…”

Section: Resultsmentioning

confidence: 95%

“…1B) established a subunit mass for the enzyme of 32 5 0.5 kDa. Uro'gen I11 synthase from B. subtilis is therefore clearly a monomeric enzyme, in common with those isolated from other sources [5,6,8,201. The predicted subunit mass for uro'gen I11 synthase from B. subtilis, calculated from the aminoacid composition derived from the DNA sequence of the hemD gene, is 29.1 kDa [lo].…”

Section: Resultsmentioning

confidence: 98%

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Expression, Purification and Characterisation of the Product from the Bacillus Subtilis hemD gene, Uroporphyrinogen III Synthase

Stamford

Capretta

Battersby

1995

European Journal of Biochemistry

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Uroporphyrinogen I11 synthase, the product of the hemD gene, is the enzyme responsible for the cyclisation of the linear tetrapyrrole, hydroxymethylbilane. The hemD gene isolated from Bacillus subtilis was manipulated by PCR to enable direct cloning behind a synthetic ribosome-binding site downstream of tandem bacteriophage A P, and PL promoters in a pCE30-derived vector. Following thermal induction of transcription, the resulting plasmid (pPS21) directed the synthesis of uroporphyrinogen 111 synthase. The protein produced was soluble and was readily purified. Pure uroporphyrinogen I11 synthase is monomeric with an isoelectric point of 4.1 and an optimum pH for activity of 8.3. Its specific activity by assay using synthetic hydroxymethylbilane as substrate is 565 units mg-' and the K , for this substrate is 330 2 30 nM. The N-terminal sequence of the enzyme is Met-Glu-Asn-Asp-Phe-Pro-Leu, in agreement with the gene-derived sequence. Studies based on amino acid modifications suggest that arginine, lysine and probably histidine residues are essential for the activity of uroporphyrinogen 111 synthase. Significantly, this synthase from B. subtilis is substantially more thermostable than the enzymes from previously studied sources.

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Purification and properties of uroporphyrinogen III synthase (co-synthase) from an overproducing recombinant strain of Escherichia coli K-12

Cited by 49 publications

References 23 publications

Unique Properties of Plasmodium falciparum Porphobilinogen Deaminase

Unique Properties of Plasmodium falciparum Porphobilinogen Deaminase

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

Expression, Purification and Characterisation of the Product from the Bacillus Subtilis hemD gene, Uroporphyrinogen III Synthase

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