Purine nucleotide biosynthesis in Escherichia coli K12 : molecular biology of the purB gene
Purine nucleotide biosynthesis in Escherichia coli K12 : molecular biology of the purB gene
The purB gene maps at 25 minutes on the Escherichia coli chromosome and codes for the bifunctional enzyme succinyl-AMP lyase (SALE) (EC 4.3.3.2). SALE catalyses two analogous fumarate elimination reactions: the conversion of 5'-phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazole to 5'-phosphoribosyl-4-carboxamide-5-aminoimidazole in the biosynthesis of AMP and GMP and the conversion of succinyl-AMP to AMP. purB was isolated from a plasmid (p124) derived from an E.coli DNA library. p124 was shown to complement a purB mutant strain and direct the synthesis of elevated levels of SALE. The purB region was cloned into the vector pUC18 giving plasmid pSG108. Strains harbouring pSG108 exhibited up to 238-fold overexpression of SALE activity. SALE levels were repressed by growth in the presence of excess adenine. SALE was purified to near-homogeneity from an overexpressing strain by anion-exchange FPLC. The purified enzyme appears to be a homotetramer of Mr about 200,000. It has a Km for succinyl-AMP of 3.7 μM, a pH optimum of 7.4 - 7.6 and is inhibited by AMP, but not at physiologically significant concentrations. Construction of overlapping nested deletions and oligonucleotide primers was used to obtain the nucleotide sequence of 2986 bp of the purB region. The purB open reading frame codes for a SALE subunit of 456 amino acids and M_r 51,542. The derived N-terminal amino acid sequence of SALE is in good agreement with that of the purified enzyme. The SALE amino acid sequence is about 24% positionally identical to that of succinyl-AMP lyase from B.subtilis and comparison with the related enzymes aspartase and fumarase (class II) from E.coli and argininosuccinate lyase from yeast revealed a highly conserved fumarate lyase motif. Downstream of purB is a probable rho-independent transcriptional terminator. The purB coding region contains a putative DnaA box and pur operator which may permit termination of transcription by DnaA protein and PurR repressor binding, respectively. The latter would account for the purine-mediated repression of SALE levels that has been observed. An unidentified open reading frame (ORF-23), coding for a 213 amino acid product of M_r 22,915, occurs 3 bp upstream of purB. A potential igma^70 promoter sequence occurs 64 bp upstream of ORF-23 suggesting that ORF-23 and purB may be cotranscribed. The derived ORF-23 amino acid sequence has the potential to form an amphipathic coiled coil motif, but closely resembles no sequenced protein. A third open reading frame of unknown expressivity and function occurs upstream of ORF-23. A partial open reading frame downstream of purB was identified as that of phoP, a transcriptional regulator in S.typhimurium. The cloned purB region was found to be lethal in certain host backgrounds. The effects of deletion plasmids on cell viability suggest that this lethality may be attributable to phoP.
University of Southampton
1992
Green, Stephen Mark
(1992)
Purine nucleotide biosynthesis in Escherichia coli K12 : molecular biology of the purB gene.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The purB gene maps at 25 minutes on the Escherichia coli chromosome and codes for the bifunctional enzyme succinyl-AMP lyase (SALE) (EC 4.3.3.2). SALE catalyses two analogous fumarate elimination reactions: the conversion of 5'-phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazole to 5'-phosphoribosyl-4-carboxamide-5-aminoimidazole in the biosynthesis of AMP and GMP and the conversion of succinyl-AMP to AMP. purB was isolated from a plasmid (p124) derived from an E.coli DNA library. p124 was shown to complement a purB mutant strain and direct the synthesis of elevated levels of SALE. The purB region was cloned into the vector pUC18 giving plasmid pSG108. Strains harbouring pSG108 exhibited up to 238-fold overexpression of SALE activity. SALE levels were repressed by growth in the presence of excess adenine. SALE was purified to near-homogeneity from an overexpressing strain by anion-exchange FPLC. The purified enzyme appears to be a homotetramer of Mr about 200,000. It has a Km for succinyl-AMP of 3.7 μM, a pH optimum of 7.4 - 7.6 and is inhibited by AMP, but not at physiologically significant concentrations. Construction of overlapping nested deletions and oligonucleotide primers was used to obtain the nucleotide sequence of 2986 bp of the purB region. The purB open reading frame codes for a SALE subunit of 456 amino acids and M_r 51,542. The derived N-terminal amino acid sequence of SALE is in good agreement with that of the purified enzyme. The SALE amino acid sequence is about 24% positionally identical to that of succinyl-AMP lyase from B.subtilis and comparison with the related enzymes aspartase and fumarase (class II) from E.coli and argininosuccinate lyase from yeast revealed a highly conserved fumarate lyase motif. Downstream of purB is a probable rho-independent transcriptional terminator. The purB coding region contains a putative DnaA box and pur operator which may permit termination of transcription by DnaA protein and PurR repressor binding, respectively. The latter would account for the purine-mediated repression of SALE levels that has been observed. An unidentified open reading frame (ORF-23), coding for a 213 amino acid product of M_r 22,915, occurs 3 bp upstream of purB. A potential igma^70 promoter sequence occurs 64 bp upstream of ORF-23 suggesting that ORF-23 and purB may be cotranscribed. The derived ORF-23 amino acid sequence has the potential to form an amphipathic coiled coil motif, but closely resembles no sequenced protein. A third open reading frame of unknown expressivity and function occurs upstream of ORF-23. A partial open reading frame downstream of purB was identified as that of phoP, a transcriptional regulator in S.typhimurium. The cloned purB region was found to be lethal in certain host backgrounds. The effects of deletion plasmids on cell viability suggest that this lethality may be attributable to phoP.
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Published date: 1992
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Local EPrints ID: 461251
URI: http://eprints.soton.ac.uk/id/eprint/461251
PURE UUID: d08ceae5-c5ca-4148-b13d-6e945d14ee48
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Date deposited: 04 Jul 2022 18:41
Last modified: 04 Jul 2022 18:41
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Author:
Stephen Mark Green
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