Evaluation of Functionally Important Amino Acids in <scp>l</scp>-Aspartate Ammonia-Lyase from <i>Escherichia coli</i>

Jayasekera, Maithri M.K.; Shi, Wuxian; Farber, Gregory K.; Viola, Ronald E.

doi:10.1021/bi970452x

Cited by 37 publications

(24 citation statements)

References 24 publications

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“…This enzyme is responsible for the formation of fumarate and ammonium ions from L-aspartate (Jayasekera et al 1997). Wolinella succinogenes uses fumarate as a sole carbon source during anaerobic respiration (Baar et al 2003), thus Wolinella succinogenes was grown with or without 0.2% ox-bile, and total RNA was extracted from cells to determine the relative expression ratio of genes based on the comparative Ct method (Livak and Schmittgen 2001).…”

Section: Discussionmentioning

confidence: 99%

Wolinella succinogenes response to ox-bile stress

Tan

Kovách

et al. 2007

Antonie van Leeuwenhoek

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The bacterium Wolinella succinogenes is the only known species of its genus. It was first isolated from cow ruminal fluid, and in cattle, it dwells in the reticulum and rumen compartments of the stomach. The global protein response of W. succinogenes to ox-bile was investigated with the aim to understand bile-tolerance mechanisms of the bacterium. Bacteria were grown in liquid media supplemented with different bile concentrations to determine its effects on growth and morphology. Proteomic analyses served to identify 14 proteins whose expression was modulated by the presence of 0.2% bile. Quantitative real-time PCR analyses of the expression of selected genes were employed to obtain independent confirmation of the proteomics data. Proteins differentially expressed revealed metabolic pathways involved in the adaptation of W. succinogenes to bile. The data suggested that bile stress elicited complex physiological responses rather than just specific pathways, and identified proteins previously unknown to be involved in the adaptation of bacteria to bile.

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Section: Discussionmentioning

confidence: 99%

Wolinella succinogenes response to ox-bile stress

Tan

Kovách

et al. 2007

Antonie van Leeuwenhoek

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“…The importance of specific amino acids within aspartases from different bacteria for substrate binding and catalytic function have been identified (10). Such characterization allows direct comparison with similar enzymes from E. corrodens and other bacteria.…”

Section: Aspartasementioning

confidence: 99%

Characterization and expression of adjacent proline iminopeptidase and aspartase genes from Eikenella corrodens

Selby

Allaker

Dymock

2003

Oral Microbiology and Immunology

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Two adjacent genes involved in nitrogen metabolism from Eikenella corrodens, with a potential role in pathogenesis, were studied. Proline iminopeptidase (Pip) activity, which may be essential for energy production and protection against host immune mechanisms, is exhibited by E. corrodens. Analysis of Pip-expressing clones revealed an ORF of 939 bases with a predicted amino acid sequence identity of 67% to the Pip of Neisseria gonorrhoea. 200 bp downstream from pip, an ORF of 1395 bases, encoding a protein with 87% identity to a putative aspartase from the Neisseria meningitidis genome sequence, was identified. Enzymatic function was confirmed with a complemented Escherichia coli aspartase deficient mutant. The E. corrodens aspartase was found to be 77% identical to the Haemophilus influenzae aspartase sequence, which was originally identified on the basis of its ability to bind plasminogen. However, the E. corrodens aspartase had no such activity. Southern hybridization indicated both genes to be single copy and conserved within the genomes of a diverse panel of E. corrodens isolates from health and disease.

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“…The standard assay system consists of 100 mM sodium L-aspartate (pH 7.0), 6 mM MgCl 2 , 100 mM Tris-HCl (pH 7.0), and the enzyme. In the assay of the fumarateamination reaction, the activity was monitored spectrophotometrically by measuring the decrease in the absorbance at 240 nm caused by the disappearance of fumarate in 1.0 ml of an assay mixture that consists of 50 mM TAPS 1 -NaOH buffer (pH 8.5), 5 mM fumarate, 100 mM NH 4 Cl, the enzyme, and various concentrations of MgCl 2 and L-aspartate.…”

Section: Cloning the Selected Genes Of Drasp Into Pet-22b(ϩ)-mentioning

confidence: 99%

“…L-Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) catalyzes the reversible conversion of L-aspartate to fumarate and NH 4 ϩ (1). L-Aspartase from Escherichia coli W is composed of four identical subunits that are arranged with the point symmetry P2 1 2 1 2 1 (2).…”

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confidence: 99%

A Monomeric l-Aspartase Obtained by in Vitro Selection

Kong

Gou

et al. 2002

Journal of Biological Chemistry

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By mimicking the partial spatial structure of the dimer of the L-aspartase subunit, the central ten-helix bundle, and an "active site" between the cleft of domain 1 (D1) and domain 3 (D3) from different subunits, we designed L-aspartase variants, in which D1D2 and D2D3 were ligated with a random hexapeptide loop. As expected, we obtained the variant with the highest activity (relative activity is 21.3% of the native enzyme, named as drAsp017) by in vitro selection. The molecular weight of this variant, obtained from size-exclusion column chromatography, is about 81 kDa, which indicates that it is indeed a monomer, whereas native L-aspartase is a tetramer. The activity-reversibility of drAsp017 (10 ؊7 M) was 80% after incubation for 30 min at 50°C, while native enzyme only retained about 17% under the same conditions. Reactivation of drAsp017 denatured in 4 M guanidine HCl was independent of protein concentration at up to 20 ؋ 10 ؊8 M at 25°C, whereas the protein concentration of native enzyme strongly affected its reactivation under the above conditions. The sensitivity of drAsp017 (10 ؊7 M) to effective factors in the fumarateamination reaction compared with native enzyme was also determined. Half-saturating concentrations of the activator L-aspartate and Mg 2؉ for drAsp017 (0.8 and 0.5 mM, respectively) are much higher than that of the native enzyme (0.10 and 0.15 mM, respectively). The data show that a monomeric L-aspartase is obtained by in vitro selection. Thus, the conversion of oligomeric proteins into their functional monomers could have important applications.L-Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) catalyzes the reversible conversion of L-aspartate to fumarate and NH 4 ϩ (1). L-Aspartase from Escherichia coli W is composed of four identical subunits that are arranged with the point symmetry P2 1 2 1 2 1 (2). The monosubunit of native L-aspartase has no catalytic function (3). Each subunit consists of three domains oriented in an elongated S-shape: D1, D2, and D3 (see Fig. 1a). The central domain (D2), containing more than half of the total residues, is the most conserved domain in the aspartase-fumatase structural family. The central core of D2 is made up of five long helices, which are all slightly bent and are nearly parallel to each other. In maintaining the active conformation, it is important to account for most of the intersubunit contacts in L-aspartase; the five-helix bundles of D2 from four subunits form a stable structure (a 20-helix cluster) in the tetramer (4) (see Fig. 1b).L-Aspartase is an important enzyme used in industry. However, the practical application for L-aspartase in aspartate synthesis has been limited, mostly due to its relatively poor stability (5). As a tetramer protein, L-aspartase dissociates easily under industrial production conditions, and this is accompanied by the loss of its catalytic activity. In the presence of 0.4 M guanidine HCl, 45% of the native activity was observed when the L-aspartase tetramer had been reversibly dissociated into dimer. Fluorescen...

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Evaluation of Functionally Important Amino Acids in l-Aspartate Ammonia-Lyase from Escherichia coli

Cited by 37 publications

References 24 publications

Wolinella succinogenes response to ox-bile stress

Wolinella succinogenes response to ox-bile stress

Characterization and expression of adjacent proline iminopeptidase and aspartase genes from Eikenella corrodens

A Monomeric l-Aspartase Obtained by in Vitro Selection

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