Identification of the gene (aphA) encoding the class B acid phosphatase/phosphotransferase of Escherichia coli MG1655 and characterization of its product

Thaller, María Cristina

doi:10.1016/s0378-1097(96)00474-0

Cited by 27 publications

(47 citation statements)

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“…AphA is an oligomeric protein made of four identical 25-kDa subunits that is secreted in the periplasmic space. The enzyme exhibits optimal activity at acidic pH values and requires a metal co-factor for activity, as suggested by susceptibility to inhibition by EDTA [2] and by crystallographic data [3].…”

Section: Introductionmentioning

confidence: 92%

“…The culture was induced with isopropyl-h-d-thiogalactopyranoside (IPTG) (0.5 mM final concentration) when A 600 reached a value of 0.5. Cells were collected by centrifugation (15,000Âg for 45 min at 4 -C) 15 h after induction, and the enzyme was extracted from the periplasmic space as described previously [2]. The enzyme was precipitated from the periplasmic extract by adding 30% (w/v) PEG6000.…”

Section: Apha Production and Purificationmentioning

confidence: 99%

“…Preliminary functional characterization showed that the enzyme is able to dephosphorylate various organic phosphomonoesters, including 5V-and 3V-mononucleotides, 2V-deoxy-5V-mononucleotides, aryl-phosphates and glycerol 2-phosphate, being also able to catalyze the transfer of phosphate from pnitrophenyl phosphate ( pNPP) to hydroxyl groups of other organic compounds [2].…”

Section: Introductionmentioning

confidence: 99%

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Biochemical characterization of the class B acid phosphatase (AphA) of Escherichia coli MG1655

Passariello

Forleo

Micheli

et al. 2006

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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The AphA enzyme of Escherichia coli, a molecular class B periplasmic phosphatase that belongs to the DDDD superfamily of phosphohydrolases, was purified and subjected to biochemical characterization. Kinetic analysis with several substrates revealed that the enzyme essentially behaves as a broad-spectrum nucleotidase highly active on 3V-and 5V-mononucleotides and monodeoxynucleotides, but not active on cyclic nucleotides, or nucleotides di-and triphosphate. Mononucleotides are degraded to nucleosides, and AphA apparently does not exhibit any nucleotide phosphomutase activity. However, it can transphosphorylate nucleosides in the presence of phosphate donors. Kinetic properties of AphA are consistent with structural data, and suggest a role for the hydrophobic pocket present in the active site crevice, made by residues Phe 56, Leu71, Trp77 and Tyr193, in conferring preferential substrate specificity by accommodating compounds with aromatic rings. AphA was inhibited by several chelating agents, including EDTA, EGTA, 1,10-phenanthroline and dipicolinic acid, with EDTA being apparently the most powerful inhibitor. D

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Section: Introductionmentioning

confidence: 92%

Section: Apha Production and Purificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Biochemical characterization of the class B acid phosphatase (AphA) of Escherichia coli MG1655

Passariello

Forleo

Micheli

et al. 2006

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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“…Contamination by cytoplasmic membranes, analysed by testing for NADPH dehydrogenase activity [20], was found to be lower than 10%. Periplasmic proteins were extracted by a spheroplasting procedure using lysozyme and EDTA, as described previously [21].…”

Section: Cell Fractionation Proceduresmentioning

confidence: 99%

“…Isopropyl h-D-thiogalactopyranoside (Sigma Chemical, St. Louis, MO, USA) (0.5 mM final concentration) was added to the culture when it reached an OD 600 of 1.5 (delayed addition of IPTG, compared to standard conditions, was found to be beneficial to sOlpA expression in preliminary experiments). Three hours after induction, cells were harvested by centrifugation, and periplasmic proteins were extracted by a spheroplasting procedure using lysozyme and EDTA [21]. The sOlpA protein was precipitated from the periplasmic extract with 30% (w/ v) polyethylene glycol (PEG) 6000 at 4 jC for 18 h. The precipitate was collected by centrifugation (45,000Âg for 2 h at 4 jC), resuspended in 30 ml of 20 mM Tris -HCl (pH 7.5), and loaded (at a flow rate of 1 ml/min) onto a DEAESepharose FF column (7 by 2.6 cm, Amersham-Pharmacia Biotech, Milan, Italy) previously equilibrated with the same buffer.…”

Section: Purification Of the Solpa Proteinmentioning

confidence: 99%

The molecular class C acid phosphatase of Chryseobacterium meningosepticum (OlpA) is a broad-spectrum nucleotidase with preferential activity on 5′-nucleotides

Passariello

Schippa

Iori

et al. 2003

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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The olpA gene of Chryseobacterium meningosepticum, encoding a molecular class C phosphatase, was cloned and expressed in Escherichia coli. The gene encodes a 29-kDa polypeptide containing an amino-terminal signal peptide typical of bacterial membrane lipoproteins. Expression in E. coli results in a functional product that mostly partitions in the outer membrane. A secreted soluble OlpA derivative (sOlpA) lacking the N-terminal cysteine residue for lipid anchoring was produced in E. coli and purified by means of two steps of ion exchange chromatography. Analysis of the kinetic parameters of sOlpA with several organic phosphoesters revealed that the enzyme was able to efficiently hydrolyze nucleotide monophosphates, with a strong preference for 5V -nucleotides and for 3V -AMP. The enzyme was also able to hydrolyze sugar phosphates and h-glycerol phosphate, although with a lower efficiency, whereas it was apparently inactive against nucleotide di-and triphosphates, diesters, and phytate. OlpA, therefore, can be considered a broad-spectrum nucleotidase with preference for 5V -nucleotides. Its functional behaviour exhibits differences from that of the Haemophilus influenzae OMP P4 lipoprotein, revealing functional heterogeneity among phosphatases of molecular class C. D

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Conserved sequence motifs among bacterial, eukaryotic, and archaeal phosphatases that define a new phosphohydrolase superfamily

1998

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Members of a new molecular family of bacterial nonspecific acid phosphatases (NSAPs), indicated as class C, were found to share significant sequence similarities to bacterial class B NSAPs and to some plant acid phosphatases, representing the first example of a family of bacterial NSAPs that has a relatively close eukaryotic counterpart. Despite the lack of an overall similarity, conserved sequence motifs were also identified among the above enzyme families (class B and class C bacterial NSAPs, and related plant phosphatases) and several other families of phosphohydrolases, including bacterial phosphoglycolate phosphatases, histidinol-phosphatase domains of the bacterial bifunctional enzymes imidazole-glycerolphosphate dehydratases, and bacterial, eukaryotic, and archaeal phosphoserine phosphatases and threalose-6-phosphatases. These conserved motifs are clustered within two domains, separated by a variable spacer region, according to the pattern [FILMAVTI-D-[ILFRMVYI-D-[GSNDEI-[TVI-[ILVAMI-[ATSVILMC]-X-{YFWHKR}-X-{YFWHNQ}-X(lO2,19l)-{KRHNQ}-G-D-{FYWHILVMC}-{QNH}-{FWYGP}-D-{PSNQYW}. The dephosphorylating activity common to all these proteins supports the definition of this phosphatase motif and the inclusion of these enzymes into a superfamily of phosphohydrolases that we propose to indicate as "DDDD' after the presence of the four invariant aspartate residues. Database searches retrieved various hypothetical proteins of unknown function containing this or similar motifs, for which a phosphohydrolase activity could be hypothesized.Keywords: acid phosphatase; homology; molecular family; molecular superfamily; phosphatase; sequence motif Phosphohydrolases (phosphatases) are enzymes that catalyze the dephosphorylation of various substrates by hydrolysis of phosphoReprint requests to:

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Identification of the gene (aphA) encoding the class B acid phosphatase/phosphotransferase of Escherichia coli MG1655 and characterization of its product

Cited by 27 publications

References 0 publications

Biochemical characterization of the class B acid phosphatase (AphA) of Escherichia coli MG1655

Biochemical characterization of the class B acid phosphatase (AphA) of Escherichia coli MG1655

The molecular class C acid phosphatase of Chryseobacterium meningosepticum (OlpA) is a broad-spectrum nucleotidase with preferential activity on 5′-nucleotides

Conserved sequence motifs among bacterial, eukaryotic, and archaeal phosphatases that define a new phosphohydrolase superfamily

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