Structure and expression in Escherichia coli K-12 of the L-asparaginase I-encoding ansA gene and its flanking regions

Jerlström, Pierre G.; Bezjak, Daniel A.; Jennings, Michael P.; Beacham, Ifor R.

doi:10.1016/0378-1119(89)90312-0

Cited by 43 publications

(39 citation statements)

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“…The amino acid sequence of L-asparaginase II has been previously determined (24). A comparison of these two sequences confirms indirect evidence (40) that they are highly dissimilar; a single region of significant homology, however, is consistent with their likely origin via an ancient gene duplication (19).…”

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confidence: 58%

“…Escherichia coli produces two L-asparaginases with markedly different properties. One enzyme, L-asparaginase I, has a low affinity for L-asparagine (Ki,, 3.5 x l0' M), is cytoplasmic, and is thought to be constitutively produced (19,50). L-Asparaginase II, by contrast, is a high-affinity enzyme (Ki,m 1.15 x 1O-5 M) and is secreted to the periplasm, and its expression is positively regulated (6,8,13,20,35).…”

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confidence: 99%

“…Molecular genetic studies have recently resulted in the isolation of the gene (ansA) encoding L-asparaginase I and hence the primary structure of this enzyme (19). The amino acid sequence of L-asparaginase II has been previously determined (24).…”

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confidence: 99%

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Analysis of the Escherichia coli gene encoding L-asparaginase II, ansB, and its regulation by cyclic AMP receptor and FNR proteins

Jennings

Beacham

1990

J Bacteriol

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Escherichia coli contains two L-asparaginase isozymes: L-asparaginase I, a low-affinity enzyme located in the cytoplasm, and L-asparaginase II, a high-affinity secreted enzyme. A molecular genetic analysis of the gene (ansA) encoding the former enzyme has previously been reported. We now present a molecular study of the gene, ansB, encoding L-asparaginase II. This gene was isolated by using oligonucleotide probes, whose sequences were based on the previously determined amino acid sequence. The nucleotide sequence of ansB, including 5'-and 3'-untranslated regions, was determined. The amino acid sequence of L-asparaginase II, deduced from this nucleotide sequence, contains differences at 11 positions when compared with the previously determined amino acid sequence. The deduced amino acid sequence also reveals a typical secretory signal peptide of 22 residues. A single region of sequence similarity is observed when ansA and ansB are compared.The transcriptional start site in ansB was determined, allowing the identification of the promoter region. The regulation of ansB was studied by using ansB'-'lacZ fusions, together with a deletion analysis of the 5' region upstream of the promoter. Regulation by cyclic AMP receptor protein and anerobiosis (FNR protein) was confirmed, and the presence of nucleotide sequence motifs, with homology to cyclic AMP receptor protein and FNR protein-binding sites, investigated.

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Analysis of the Escherichia coli gene encoding L-asparaginase II, ansB, and its regulation by cyclic AMP receptor and FNR proteins

Jennings

Beacham

1990

J Bacteriol

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“…His1 97 is conserved in three high-affinity Alignments were made using the program MACAW [21]. EcoA2, E. coli asparaginase IT [22]; AgGA, Acinetabacter glutaminas$icans glutaminase-asparaginase [23] ; ErcA, asparaginase from Erwinia chrysanthemi [24]; BsuA, asparaginase from Bacillus subtilis [25]; YeaA2, Yeast asparaginase I1 [26]; EcoAl , E. coli asparaginase I [27]. Residues appearing in three or more of the sequences are shown in capitals, conservative replacements are indicated by italics.…”

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Site‐specific mutagenesis of Escherichia coli asparaginase II

Wehner

Harms

Jennings

et al. 1992

European Journal of Biochemistry

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Site-specific mutagenesis was used to replace the three histidine residues of Escherichiu coli asparaginase I1 (EcA2) with other amino acids. The following enzyme variants were studied: H87KIEcA2, IH183LIEcA2 and [H197L]EcM. None of the mutations substantially affected the K , for L-aspartic acid fl-hydroxamate or impaired aspartate binding. The relative activities towards L-Asn, L-Gln, and 1-aspartic acid 8-hydroxamate were reduced to the same extent, with residual activities exceeding 10% of the wild-type values. These data do not support a number of previous reports suggesting that histidine residues are essential for catalysis.

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“…R. etli asparaginase is found to be positively regulated by its substrate asparagine and negatively regulated by the carbon source; it is not regulated by the amount of oxygen dissolved in the growth medium or by nitrogen catabolite repression, and some asparaginase activity is detected when R. etli is grown on ammonium as a nitrogen source and succinate as a carbon source (20). Asparaginase has been studied in other gramnegative bacteria such as Escherichia coli (10,12,22,24,25,37,47), Salmonella enterica (23), Erwinia chrysanthemi (19), and Vibrio proteus (41) and in gram-positive bacteria such as Bacillus subtilis (1,21,43) and Staphylococcus aureus (35,42).…”

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Isolation and characterization of Rhizobium etli mutants altered in degradation of asparagine

et al. 1997

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Rhizobium etli mutants unable to grow on asparagine as the nitrogen and carbon source were isolated. Two kinds of mutants were obtained: AHZ1, with very low levels of aspartase activity, and AHZ7, with low levels of asparaginase and very low levels of aspartase compared to the wild-type strain. R. etli had two asparaginases differentiated by their thermostabilities, electrophoretic mobilities, and modes of regulation. The AHZ mutants nodulated as did the wild-type strain and had nitrogenase levels similar to that of the wild-type strain.Amino acid synthesis has been studied in various microorganisms; amino acid degradation has been less studied, despite the fact that the intracellular amino acid concentration must be the result of its synthesis and degradation. In Neurospora crassa and Rhizobium etli, it has been shown that the intracellular glutamine concentration is the result not only of the regulation of the synthesis but also of the degradation of this amino acid (4, 6-8, 15, 16, 31). To determine if the intracellular concentrations of other amino acids are regulated in the same way as that of glutamine, we decided to study the degradation of asparagine (an amino acid very similar to glutamine, with four carbon skeletons instead of five) in R. etli, a bacterium that establishes symbiosis with Phaseolus vulgaris (39).In R. etli, L-asparagine can be utilized as the sole carbon and nitrogen source through the action of two enzymes (20). The first enzyme, asparaginase (EC 3.5.1.1), catalyzes the hydrolysis of L-asparagine to L-aspartate and ammonium. The second enzyme, L-aspartase (EC 4.3.1.1), catalyzes the reversible deamination of L-aspartate to yield fumarate and ammonium. R. etli asparaginase is found to be positively regulated by its substrate asparagine and negatively regulated by the carbon source; it is not regulated by the amount of oxygen dissolved in the growth medium or by nitrogen catabolite repression, and some asparaginase activity is detected when R. etli is grown on ammonium as a nitrogen source and succinate as a carbon source (20). Asparaginase has been studied in other gramnegative bacteria such as Escherichia coli (10,12,22,24,25,37,47), Salmonella enterica (23), Erwinia chrysanthemi (19), and Vibrio proteus (41) and in gram-positive bacteria such as Bacillus subtilis (1, 21, 43) and Staphylococcus aureus (35,42).R. etli aspartase was found to be positively regulated by asparagine, negatively regulated by the carbon source and by ammonium, and not regulated by the amount of oxygen dissolved in the growth medium (20). L-Aspartase has been studied in other bacteria such as E. coli (25,27,33,36), B. subtilis (43), Pseudomonas fluorescens (30, 45), and Serratia marcescens (44).In this report, we describe the isolation and characterization of R. etli mutants altered in the degradation of asparagine, present evidence that R. etli has two asparaginases, and investigate the regulation of these isoenzymes. The cloning of an R. etli DNA fragment that complements the isolated mutants is also reported...

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Structure and expression in Escherichia coli K-12 of the L-asparaginase I-encoding ansA gene and its flanking regions

Cited by 43 publications

References 40 publications

Analysis of the Escherichia coli gene encoding L-asparaginase II, ansB, and its regulation by cyclic AMP receptor and FNR proteins

Analysis of the Escherichia coli gene encoding L-asparaginase II, ansB, and its regulation by cyclic AMP receptor and FNR proteins

Site‐specific mutagenesis of Escherichia coli asparaginase II

Isolation and characterization of Rhizobium etli mutants altered in degradation of asparagine

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