Purification and properties of uroporphyrinogen III synthase (co-synthetase) from <i>Euglena gracilis</i>

Hart, G J; Battersby, Alan R.

doi:10.1042/bj2320151

Cited by 73 publications

(32 citation statements)

References 34 publications

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“…That the molecular mass obtained from gel filtration appeared larger than the values obtained from SDS/ PAGE and by calculation was not surprising. Similar differences have also been observed for the derived molecular masses of uro'gen 111 synthase enzymes from Euglena gracilis [5] and rat liver [20] which are probably due to substantially unsymmetrical shapes for these monomeric enzymes.…”

Section: Resultssupporting

confidence: 61%

“…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%

“…Uro'gen I11 synthase was detected and relative amounts of the enzyme were determined using a coupled assay in which the enzyme HMB synthase is used to generate the substrate HMB essentially as described [5]. Uro'gen I11 synthase was assayed quantitatively and kinetic assays were performed by monitoring uro'gen I11 formation with synthetic HMB as substrate [14].…”

Section: Methodsmentioning

confidence: 99%

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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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Section: Resultssupporting

confidence: 61%

Section: Resultsmentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

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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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“…Chemical modification studies of UrolIl synthase from E. gracilis suggest that arginine residues are essential for the activity of the enzyme (23 (11), E. coli (Eco) (31), and B. subtilis (Bsu) PBG synthase. Positions with identical amino acid residues in all five polypeptides are marked by asterisks.…”

Section: 1mentioning

confidence: 99%

The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III

et al. 1991

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We have recently reported (M. Petricek, L. Rutberg, I. Schröder, and L. Hederstedt, J. Bacteriol. 172: 2250-2258, 1990) the cloning and sequence of a Bacillus subtilis chromosomal DNA fragment containing hemA proposed to encode the NAD(P)H-dependent glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid (ALA) synthesis, hemX encoding a hydrophobic protein of unknown function, and hemC encoding hydroxymethylbilane synthase. In the present communication, we report the sequences and identities of three additional hem genes located immediately downstreatm of hemC, namely, hemD encoding uroporphyrinogen III synthase, hemB encoding porphobilinogen synthase, and hemL encoding glutamate-1-semialdehyde 2,1-aminotransferase. The six genes are proposed to constitute a hem operon encoding enzymes required for the synthesis of uroporphyrinogen III from glutamyl-tRNA. hemA, hemB, hemC, and hemD have all been shown to be essential for heme synthesis. However, deletion of an internal 427-bp fragment of hemL did not create a growth requirement for ALA or heme, indicating that formation of ALA from glutamate-1-semialdehyde can occur spontaneously in vivo or that this reaction may also be catalyzed by other enzymes. An analysis of B. subtilis carrying integrated plasmids or deletions-substitutions in or downstream of hemL indicates that no further genes in heme synthesis are part of the proposed hem operon.

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“…It is now recognized that HMB-synthase catalyzes the head to tail condensation offour molecules of porphobilinogen to form the linear tetrapyrrole HMB (4,5). In the presence of URO-synthase, HMB is rapidly converted to uroporphyrinogen III by an intramolecular rearrangement of the D-pyrrole group and ring closure (5)(6)(7). In the absence of URO-synthase, HMB nonenzymatically cyclizes to form the nonphysiologic uroporphyrinogen I isomer.…”

mentioning

confidence: 99%

Human uroporphyrinogen III synthase: molecular cloning, nucleotide sequence, and expression of a full-length cDNA.

Tsai

Bishop

Desnick

1988

Proc. Natl. Acad. Sci. U.S.A.

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Uroporphyrinogen III synthase [URO-synthase; hydroxymethylbilane hydro-lyase (cyclizing), EC 4.2.1.75], the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 x 106 recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The authenticity of this clone was established by colinearity of the predicted amino acid sequence with 81 microsequenced residues from the purified enzyme. In addition, high levels of enzymatic activity and immunoreactive protein were expressed when a blunt-ended 971-base-pair Ava II cDNA fragment containing the entire coding region was inserted into vectors for expression in Escherichia coli. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria. were required for the conversion of the monopyrrole, porphobilinogen, to the cyclic tetrapyrrole, uroporphyrinogen III (2). During the next two decades, the precise function of the two enzymes in uroporphyrinogen III biosynthesis was the subject of active investigation and debate, particularly with regard to their reaction mechanisms and intermediates and their potential interaction in a cytosolic enzyme complex (for reviews, see refs. 3 and 4). It is now recognized that HMB-synthase catalyzes the head to tail condensation offour molecules of porphobilinogen to form the linear tetrapyrrole HMB (4, 5). In the presence of URO-synthase, HMB is rapidly converted to uroporphyrinogen III by an intramolecular rearrangement of the D-pyrrole group and ring closure (5-7). In the absence of URO-synthase, HMB nonenzymatically cyclizes to form the nonphysiologic uroporphyrinogen I isomer.The deficient activity of URO-synthase is the enzymatic defect in congenital erythropoietic porphyria, an inborn error of heme biosynthesis that is inherited as an autosomal recessive trait (8). Enzymatic diagnosis of affected homozygotes and asymptomatic heterozygous carriers can be made by the demonstration of deficient or intermediate lev...

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Purification and properties of uroporphyrinogen III synthase (co-synthetase) from Euglena gracilis

Cited by 73 publications

References 34 publications

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

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

The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III

Human uroporphyrinogen III synthase: molecular cloning, nucleotide sequence, and expression of a full-length cDNA.

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