Purification and characterization of alkaline phosphatase containing phosphotyrosyl phosphatase activity from the bacterium <i>Prevotella intermedia</i>

Ansai, Toshihiro; Awano, Shuji; Chen, Xiaochi; Fuchi, Tomoko; Arimoto, Takafumi; Akifusa, Sumio; Takehara, Tadamichi

doi:10.1016/s0014-5793(98)00514-6

Cited by 20 publications

(14 citation statements)

References 18 publications

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“…Vanadate appeared to be a weak inhibitor of P. abyssi AP. This is not consistent with previous studies on other APs (1,6,21) and may be a result of the experimental conditions (pH 10.0, 70°C) or of an original catalytic mechanism. In contrast, thiol-reducing agents exhibited a strong inhibitory effect on AP activity.…”

Section: Discussioncontrasting

confidence: 99%

Characterization of a Highly Thermostable Alkaline Phosphatase from the EuryarchaeonPyrococcus abyssi

Zappa

Rolland

Flament

et al. 2001

Appl Environ Microbiol

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This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon. An AP gene from Pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in Escherichia coli. Analysis of the sequence showed conservation of the active site and structural elements of the E. coli AP. The recombinant AP was purified and characterized. Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa. Apparent optimum pH and temperature were estimated at 11.0 and 70°C, respectively. Thus far, P. abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105°C of 18 and 5 h, respectively. Enzyme activity was found to be dependent on divalent cations: metal ion chelators inhibited activity, whereas the addition of exogenous Mg(II), Zn(II), and Co(II) increased activity. The enzyme was inhibited by inorganic phosphate, but not by molybdate and vanadate. Strong inhibitory effects were observed in the presence of thiolreducing agents, although cysteine residues of the P. abyssi AP were not found to be incorporated within intraor interchain disulfide bonds. In addition, P. abyssi AP was demonstrated to dephosphorylate linear DNA fragments with dephosphorylation efficiencies of 93.8 and 84.1% with regard to cohesive and blunt ends, respectively.

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Section: Discussioncontrasting

confidence: 99%

Characterization of a Highly Thermostable Alkaline Phosphatase from the EuryarchaeonPyrococcus abyssi

Zappa

Rolland

Flament

et al. 2001

Appl Environ Microbiol

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“…Although the exact role of oral microbes in the etiology of periodontal disease remains unknown, they have been shown to produce a variety of potential virulence factors, including high phosphatase activity (38). To date, alkaline phosphatases (ALPases) of both P. intermedia and Porphyromonas gingivalis have been characterized (3,48). A neutral phosphatase gene was also isolated from the oral spirochete Treponema denticola, which is associated with chronic periodontal disease (14).…”

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confidence: 99%

“…For instance, small, acidic PTPases contain N-terminally located, highly conserved active sites (FVCXGN ICRSPXAEAXF) (20). Moreover, PiALP, an ALPase from P. intermedia, appeared to represent a new family of alkaline PTPases that showed significant homology with the predicted primary structures of PhoD from Zymomonas mobilis and PhoV from Synechococcus spp., although PTPase activities of PhoD and PhoV were not investigated (3).…”

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Isolation, Cloning, and Expression of an Acid Phosphatase Containing Phosphotyrosyl Phosphatase Activity from Prevotella intermedia

et al. 1999

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A novel acid phosphatase containing phosphotyrosyl phosphatase (PTPase) activity, designated PiACP, from Prevotella intermedia ATCC 25611, an anaerobe implicated in progressive periodontal disease, has been purified and characterized. PiACP, a monomer with an apparent molecular mass of 30 kDa, did not require divalent metal cations for activity and was sensitive to orthovanadate but highly resistant to okadaic acid. The enzyme exhibited substantial activity against tyrosine phosphate-containing peptides derived from the epidermal growth factor receptor. On the basis of N-terminal and internal amino acid sequences of purified PiACP, the gene coding for PiACP was isolated and sequenced. The PiACP gene consisted of 792 bp and coded for a basic protein with an M r of 29,164. The deduced amino acid sequence exhibited striking similarity (25 to 64%) to those of members of class A bacterial acid phosphatases, including PhoC of Morganella morganii, and involved a conserved phosphatase sequence motif that is shared among several lipid phosphatases and the mammalian glucose-6-phosphatases. The highly conservative motif HCXAGXXR in the active domain of PTPase was not found in PiACP. Mutagenesis of recombinant PiACP showed that His-170 and His-209 were essential for activity. Thus, the class A bacterial acid phosphatases including PiACP may function as atypical PTPases, the biological functions of which remain to be determined.

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“…Moreover the identified peptide shares identity with several alkaline phosphatases in N. crassa and S. commune. It has been reported that alkaline phosphatase might dephosphorylate phosphotyrosyl proteins, 28) but it is known that alkaline phosphatase activity is completely inhibited by EDTA and is increased by Ca 2þ , Mg 2þ , and Co 2þ . [28][29][30][31] The fact that EDTA, Mg 2þ , Ca 2þ , and Co 2þ at concentrations from 0.5 mM to 20 mM did not affect dephosphorylation activity in our experiments disproves that the extracellular enzyme is an alkaline phosphatase.…”

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of a Novel Thermostable Extracellular Protein Tyrosine Phosphatase fromMetarhizium anisopliaeStrain CQMa102

Wang

Peng

et al. 2006

Bioscience, Biotechnology, and Biochemistry

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Purification and characterization of alkaline phosphatase containing phosphotyrosyl phosphatase activity from the bacterium Prevotella intermedia

Cited by 20 publications

References 18 publications

Characterization of a Highly Thermostable Alkaline Phosphatase from the EuryarchaeonPyrococcus abyssi

Characterization of a Highly Thermostable Alkaline Phosphatase from the EuryarchaeonPyrococcus abyssi

Isolation, Cloning, and Expression of an Acid Phosphatase Containing Phosphotyrosyl Phosphatase Activity from Prevotella intermedia

Purification and Characterization of a Novel Thermostable Extracellular Protein Tyrosine Phosphatase fromMetarhizium anisopliaeStrain CQMa102

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