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1.
Anna Caselli Luigia Pazzagli Paolo Paoli Giampaolo Manao Guido Camici Gianni Cappugi Giampietro Ramponi 《Journal of Protein Chemistry》1994,13(1):107-115
Porcine low Mr phosphotyrosine protein phosphatase has been purified and the complete amino acid sequence has been determined. Both enzymic and chemical cleavages are used to obtain protein fragments. FAB mass spectrometry and enzymic subdigestion followed by Edman degradation have been used to determine the structure of the NH2-terminal acylated tryptic peptide. The enzyme consists of 157 amino acid residues, is acetylated at the NH2-terminus, and has arginine as COOH-terminal residue. It shows kinetic parameters very similar to other known low Mr PTPases. This PTPase is strongly inhibited by pyridoxal 5-phosphate (K=21M) like the low Mr PTPases from bovine liver, rat liver (AcP2 isoenzyme), and human erythrocyte (Bslow isoenzyme). The comparison of the 40–73 sequence with the corresponding sequence of other low Mr PTPases from different sources demonstrates that this isoform is highly homologous to the isoforms mentioned above, and shows a lower homology degree with respect to rat AcP1 and human Bfast isoforms. A classification of low Mr PTPase isoforms based on the type-specific sequence and on the sensitivity to pyridoxal 5-phosphate inhibition has been proposed.Abbreviations used PTPase
phosphotyrosine protein phosphatase
- TFA
trifluoroacetic acid
- SDS
sodium dodecylsulfate
- T
tryptic peptides
- SP
endoproteinase Glu-C peptides
- FAB
fast atom bombardment
- Ac
acetyl
- HPLC
high-performance liquid chromatography
- OPA
o-phtaldialdehyde
- PMSF
phenylmethylsulfonyl fluoride
- CD45
leukocyte common-antigen PTPase
- LAR
leukocyte-antigen-related PTPase
- PTP IB
human placental PTPase 相似文献
2.
Giampaolo Manao Luigia Pazzagli Paolo Cirri Anna Caselli Guido Camici Gianni Cappugi Ahmad Saeed Giampietro Ramponi 《The protein journal》1992,11(3):333-345
Two lowM r phosphotyrosine protein phosphatases have been isolated from rat liver. The enzymes were previously known as lowM r acid phosphatases, but several recent studies have demonstrated that this family of enzymes possesses specific phosphotyrosine protein phosphatase activity. We determined the complete amino acid sequences of the two isoenzymes and named them AcP1 and AcP2. Both consist of 157 amino acid residues, are acetylated at the NH2-terminus, and have His as the COOH-terminus. The molecular weights calculated from the sequences are 18,062 for AcP1 and 17,848 for AcP2. They are homologous except in the 40–73 zone, where about 50% of residues are different. This fact suggests that the two isoenzymes are produced by an alternative splicing mechanism. There is no homology between these two isoenzymes and the receptor-like phosphotyrosine protein phosphatases LAR, CD45, human placenta PTPase 1B, and rat brain PTPase-1. AcP1 and AcP2 are also distinct from rat liver PTPase-1 and PTPase-2, since these last enzymes have higher molecular weights. AcP1 differs from AcP2 with respect to (1) substrate affinity and (2) its sensitivity to activators and inhibitors, thus suggesting a their different physiological function. 相似文献
3.
Anna Caselli Luigia Pazzagli Paolo Paoli Giampaolo Manao Guido Camici Gianni Cappugi Giampietro Ramponi 《The protein journal》1994,13(1):107-115
Porcine low Mr phosphotyrosine protein phosphatase has been purified and the complete amino acid sequence has been determined. Both enzymic and chemical cleavages are used to obtain protein fragments. FAB mass spectrometry and enzymic subdigestion followed by Edman degradation have been used to determine the structure of the NH2-terminal acylated tryptic peptide. The enzyme consists of 157 amino acid residues, is acetylated at the NH2-terminus, and has arginine as COOH-terminal residue. It shows kinetic parameters very similar to other known low Mr PTPases. This PTPase is strongly inhibited by pyridoxal 5′-phosphate (K=21ΜM) like the low Mr PTPases from bovine liver, rat liver (AcP2 isoenzyme), and human erythrocyte (Bslow isoenzyme). The comparison of the 40–73 sequence with the corresponding sequence of other low Mr PTPases from different sources demonstrates that this isoform is highly homologous to the isoforms mentioned above, and shows a lower homology degree with respect to rat AcP1 and human Bfast isoforms. A classification of low Mr PTPase isoforms based on the type-specific sequence and on the sensitivity to pyridoxal 5?-phosphate inhibition has been proposed. 相似文献
4.
Partanen SE 《Journal of molecular histology》2008,39(2):143-152
Estrogen-induced autocrine and paracrine growth factors are thought to stimulate endometrial proliferation. However, the proliferation
is arrested at an early secretory phase although the amount of growth factors and their receptors remains constant. These
receptors are protein tyrosine kinases which cause activating receptor autophosphorylation and phosphorylation of signalling
substances. One inhibitory mechanism is the reverse dephosphorylation by phosphatases hydrolysing phosphotyrosines. Previously,
an acid phosphotyrosine phosphatase activity was found in endometrial secretory glands. The purpose of this study was to evaluate
its characteristics. Catalytic and immunohistochemical techniques were applied on sections obtained from human endometrium
and other tissues. Endometrial acid phosphatase hydrolysed phosphotyrosine, not only at acid, but also at neutral pH values.
An alternative substrate was α-naphthyl phosphate or β-glycerophosphate but not phosphoserine. Activities were inhibited by
tartrate and fluoride but not by formaldehyde. These catalytic properties are identical only to those of prostatic acid phosphatase
(PAP). A PAP-like nature was also proved by positive PAP immunohistochemistry. In conclusion, endometrial glands contain a
phosphotyrosine phosphatase which is identical to PAP. Its activity is menstrual-cycle-dependent, being present only at the
secretory phase, and it may counterbalance receptor tyrosine kinases terminating glandular proliferation despite constant
levels of growth factors and their receptors. 相似文献
5.
A low molecular weight acid phosphatase was purified to homogeneity from chicken heart with a specific activity of 42 U/mg
and a recovery of about 1%. Nearly 800 fold purification was achieved. The molecular weight was estimated to be 18 kDa by
SDS-polyacrylamide gel electrophoresis. Para-nitrophenyl phosphate, phenyl phosphate and flavin mononucleotide were efficiently hydrolysed by the enzyme and found to
be good substrates. Fluoride and tartrate had no inhibitory effect while phosphate, vanadate and molybdate strongly inhibited
the enzyme. The acid phosphatase was stimulated in the presence of glycerol, ethylene glycol, methanol, ethanol and acetone,
which reflected the phosphotransferase activity. When phosphate acceptors such as ethylene glycol concentrations were increased,
the ratio of phosphate transfer to hydrolysis was also increased, demonstrating the presence of a transphosphorylation reaction
where an acceptor can compete with water in the rate limiting step involving hydrolysis of a covalent phospho enzyme intermediate.
Partition experiments carried out with two substrates, para-nitrophenyl phosphate and phenyl phosphate, revealed a constant product ratio of 1.7 for phosphotransfer to ethylene glycol
versus hydrolysis, strongly supporting the existence of common covalent phospho enzyme intermediate. A constant ratio of K
cat/K
m, 4.3×104, found at different ethylene glycol concentrations, also supported the idea that the rate limiting step was the hydrolysis
of the phospho enzyme intermediate. 相似文献
6.
Hanan Dahche AbdulShakur Abdullah M. Ben Potters Peter J. Kennelly 《Extremophiles : life under extreme conditions》2009,13(2):371-377
The genomes of virtually all free-living archaeons encode one or more deduced protein-serine/threonine/tyrosine kinases belonging
to the so-called eukaryotic protein kinase superfamily. However, the distribution of their cognate protein-serine/threonine/phosphatases
displays a mosaic pattern. Thermoplasma volcanium is unique among the Archaea inasmuch as it is the sole archaeon whose complement of deduced phosphoprotein phosphatases includes a member of the PPM-family
of protein-serine/threonine phosphatases—a family that originated in the Eucarya. A recombinant version of this protein, TvnPPM, exhibited protein-tyrosine phosphatase in addition to its predicted protein-serine/threonine
phosphatase activity in vitro. TvnPPM is the fourth member of the PPM-family shown to exhibit such dual-specific capability, suggesting that the ancestral
versions of this enzyme exhibited broad substrate specificity. Unlike most other archeaons, the genome of T. volcanium lacks open reading frames encoding stereotypical protein-tyrosine phosphatases. Hence, the dual-specificity of TvnPPM may
account for its seemingly aberrant presence in an archaeon. 相似文献
7.
Summary Protein phosphatase 2A1 was purified from rat skeletal muscle and used to produce antisera to the three subunits of the holoenzyme. Affinity purified antibodies specific for the subunits of the phosphatase enzyme were found to recognize the type 2A1 and 2A2 phosphatase from rat skeletal muscle, heart, liver, brain and erythrocytes and were used to investigate the effects of diabetes on the levels of this enzyme in liver and heart. Phosphorylase phosphatase assays coupled with immunoblot analysis of fractionated rat liver and heart cytosol from normal and diabetic animals show no apparent differences in the quantity or activity of these enzymes following the induction of alloxan diabetes. When considering these results and the normal physiological concentrations of known effectors of these enzymes, it is likely that protein phosphatase 2A1 and 2A2 are not responsible for the dephosphorylation of phosphorylase a under physiological conditions. 相似文献
8.
The majora2–6 sialoglycoproteins in detergent-extracts of Kurloff cells were purified by anion-exchange andSambucus nigra agglutinin-affinity chromatographies. The similar ultrastructural localisations of (1)S. nigra agglutinin-gold conjugates and (2) acid phosphatase activities on the Kurloff body and particularly on its myelin figures indicated that the majora2-6 sialoglycoproteins of the Kurloff cell had acid phosphatase activity. Two-dimensional electrophoresis showed that these tartrate-sensitive phosphatases corresponded to 2 acidic (pI 3.4–3.7) polypeptides of 36 and 34 kDa. Hydrolysis with peptide-N-glycosidases F gave a 33 kDa apoprotein rich in alanine, glutamic acid, tyrosine and lysin. A lectin-affinity study demonstrated that they contained hybrid type bisected and fucosylatedN-linked oligosaccharides. Cytotoxic properties were previously attributed to Kurloff cells and other studies suggested that not only acid phosphatases but alsoa2-6-linked sialic acid residues themselves may participate in natural killer activity. 相似文献
9.
Satoshi Ugi Hiroshi Maegawa Jerrold M. Olefsky Yukio Shigeta Atsunori Kashiwagi 《FEBS letters》1994,340(3):216-220
To clarify the role of protein tyrosine phosphatase containing Src homology 2 (SH2) regions on insulin signaling, we investigated the interactions among the insulin receptor, a pair of SH2 domains of SH-PTP2 coupled to glutathione-S-transferase (GST) and insulin receptor substrate-1 (IRS-1)-GST fusion proteins (amino-portion, IRS-1N; carboxyl portion, IRS-1C). GST-SH2 protein of SH-PTP2 bound to the wild type insulin receptor, but not to that with a carboxyl-terminal mutation (Y/F2). Furthermore, even though Y/F2 receptors were used, the SH2 protein was also co-immunoprecipitated with IRS-1C, but not with IRS-1N. These results indicate that SH2 domains of SH-PTP2 can directly associate with the Y1322TXM motif on the carboxyl terminus of insulin receptors and also may bind to the carboxyl portion of IRS-1, possibly via the V1172IDL motif in vitro. 相似文献
10.
Fengzhi Yang Fangzhou Xie Ying Zhang Yu Xia Wenlu Liu Faqin Jiang Celine Lam Yixue Qiao Dongsheng Xie Jianqi Li Lei Fu 《Bioorganic & medicinal chemistry letters》2017,27(10):2166-2170
Known PTP1B inhibitors with bis-anionic moieties exhibit potent inhibitory activity, good selectivity, however, they are incapable of penetrating cellular membranes. Based upon our finding of a new pharmacophoric group in inhibition of PTP1B and the structural characteristics of the binding pocket of PTP1B, a series of bis-arylethenesulfonic acid ester derivatives were designed and synthesized. These novel molecules, particularly Y-shaped bis-arylethenesulfonic acid ester derivatives, exhibited high PTP1B inhibitory activity, moderate selectivity, and great potential in penetrating cellular membranes (compound 7p, CLog P = 9.73, Papp = 9.6 × 10-6 cm/s; IC50 = 140, 1290 and 920 nM on PTP1B, TCPTP and SHP2, respectively). Docking simulations suggested that these Y-shaped inhibitors might interact with multiple secondary binding sites in addition to the catalytic site of PTP1B. 相似文献
11.
The survival protein E (SurE) family was discovered by its correlation to stationary phase survival of Escherichia coli and various repair proteins involved in sustaining this and other stress-response phenotypes. In order to better understand this ancient and well-conserved protein family, we have determined the 2.0A resolution crystal structure of SurEalpha from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum (Pae). This first structure of an archaeal SurE reveals significant similarities to and differences from the only other known SurE structure, that from the eubacterium Thermatoga maritima (Tma). Both SurE monomers adopt similar folds; however, unlike the Tma SurE dimer, crystalline Pae SurEalpha is predominantly non-domain swapped. Comparative structural analyses of Tma and Pae SurE suggest conformationally variant regions, such as a hinge loop that may be involved in domain swapping. The putative SurE active site is highly conserved, and implies a model for SurE bound to a potential substrate, guanosine-5'-monophosphate (GMP). Pae SurEalpha has optimal acid phosphatase activity at temperatures above 90 degrees C, and is less specific than Tma SurE in terms of metal ion requirements. Substrate specificity also differs between Pae and Tma SurE, with a more specific recognition of purine nucleotides by the archaeal enzyme. Analyses of the sequences, phylogenetic distribution, and genomic organization of the SurE family reveal examples of genomes encoding multiple surE genes, and suggest that SurE homologs constitute a broad family of enzymes with phosphatase-like activities. 相似文献
12.
Hirotaka Takahashi Akihiko Ozawa Keiichirou Nemoto Akira NozawaMotoaki Seki Kazuo ShinozakiHiroyuki Takeda Yaeta Endo Tatsuya Sawasaki 《FEBS letters》2012,586(19):3134-3141
Plant genome possesses over 100 protein phosphatase (PPase) genes that are key regulators of signal transduction via phosphorylation/dephosphorylation event. Here we report a comprehensive functional analysis of protein serine/threonine, dual-specificity and tyrosine phosphatases using recombinant PPases produced by wheat cell-free protein synthesis system. Eighty-two recombinant PPases were successfully produced using Arabidopsis full-length cDNA as templates. In vitro PPase assay was performed using phosphorylated myelin basic protein as substrate. Among the AtPPases examined, 26 serine/threonine, three dual-specificity and one tyrosine PPases exhibited catalytic activity, including 20 serine/threonine and one dual-specificity PPases that showed in vitro activities for the first time. Our study demonstrates genome-wide biochemical analysis of AtPPases using wheat cell-free system, and provides new information and insights on enzyme activities.
Structured summary of protein interactions
PTP1dephosphorylatesMBP by phosphatase assay (View interaction).AtPP2CdephosphorylatesMBP by phosphatase assay (View interaction).POLTEdephosphorylatesMBP by phosphatase assay (View interaction).TOPP8dephosphorylatesMBP by phosphatase assay (View interaction).HAB1dephosphorylatesMBP by phosphatase assay (View interaction).ABI2dephosphorylatesMBP by phosphatase assay (View interaction).At1g34750dephosphorylatesMBP by phosphatase assay (View interaction).At1g43900dephosphorylatesMBP by phosphatase assay (View interaction).At3g15260dephosphorylatesMBP by phosphatase assay (View interaction).At5g53140dephosphorylatesMBP by phosphatase assay (View interaction).At1g18030dephosphorylatesMBP by phosphatase assay (View interaction).At3g06270dephosphorylatesMBP by phosphatase assay (View interaction).At2g25070dephosphorylatesMBP by phosphatase assay (View interaction).At3g02750dephosphorylatesMBP by phosphatase assay (View interaction).At5g10740dephosphorylatesMBP by phosphatase assay (View interaction).at4g26080dephosphorylatesMBP by phosphatase assay (View interaction).At4g28400dephosphorylatesMBP by phosphatase assay (View interaction).At5g06750dephosphorylatesMBP by phosphatase assay (View interaction).At4g31860dephosphorylatesMBP by phosphatase assay (View interaction).At3g17250dephosphorylatesMBP by phosphatase assay (View interaction).At4g38520dephosphorylatesMBP by phosphatase assay (View interaction).At3g05640dephosphorylatesMBP by phosphatase assay (View interaction).At5g66080dephosphorylatesMBP by phosphatase assay (View interaction).At1g79630dephosphorylatesMBP by phosphatase assay (View interaction).At2g30170dephosphorylatesMBP by phosphatase assay (View interaction).At5g24940dephosphorylatesMBP by phosphatase assay (View interaction). 相似文献13.
Subbiah Pugazhenthi Feridoon Tanha Bruce Dahl Ramji L. Khandelwal 《Molecular and cellular biochemistry》1995,153(1-2):125-129
The inhibitory action of vanadate towards protein tyrosine phosphatase (PTPase) has been considered as a probable mechanism by which it exerts insulin-like effects. In this study, we have examined thein vivo effects of vanadate on PTPases in the liver of obese Zucker rats, a genetic animal model for obesity and type II diabetes. These animals were characterized by hyperinsulinemia and mild hyperglycemia. The number of insulin receptors were significantly (p<0.01) decreased in liver. After chronic administration of vanadate in obese rats, 80% decrease in the plasma levels of insulin was observed. The insulin receptor numbers were significantly (p<0.01) higher in vanadate-treated obese rats as compared to the untreated ones. The hepatic PTPase activities in cytosolic and particulate fractions, with phosphorylated poly glu:tyr (41) and the insulin receptor peptide (residues 1142–1153) as substrates, increased in obese rats. In vanadate-treated obese rat livers, the PTPase activities in both subcellular fractions with these substrates decreased significantly (p<0.001). The decreases in PTPase activities from these groups of rats were further supported by chromatography on a Mono Q column. These data support the view that inhibition of PTPases plays a role in the insulin-mimetic action of vanadate. 相似文献
14.
Autophosphorylation of ataxia-telangiectasia mutated is regulated by protein phosphatase 2A 总被引:1,自引:0,他引:1
Goodarzi AA Jonnalagadda JC Douglas P Young D Ye R Moorhead GB Lees-Miller SP Khanna KK 《The EMBO journal》2004,23(22):4451-4461
Ionizing radiation induces autophosphorylation of the ataxia-telangiectasia mutated (ATM) protein kinase on serine 1981; however, the precise mechanisms that regulate ATM activation are not fully understood. Here, we show that the protein phosphatase inhibitor okadaic acid (OA) induces autophosphorylation of ATM on serine 1981 in unirradiated cells at concentrations that inhibit protein phosphatase 2A-like activity in vitro. OA did not induce gamma-H2AX foci, suggesting that it induces ATM autophosphorylation by inactivation of a protein phosphatase rather than by inducing DNA double-strand breaks. In support of this, we show that ATM interacts with the scaffolding (A) subunit of protein phosphatase 2A (PP2A), that the scaffolding and catalytic (C) subunits of PP2A interact with ATM in undamaged cells and that immunoprecipitates of ATM from undamaged cells contain PP2A-like protein phosphatase activity. Moreover, we show that IR induces phosphorylation-dependent dissociation of PP2A from ATM and loss of the associated protein phosphatase activity. We propose that PP2A plays an important role in the regulation of ATM autophosphorylation and activity in vivo. 相似文献
15.
Yoshiko Banno Yoshinori Nozawa 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,799(1):20-28
An extracellular acid phosphatase secreted into the medium during growth of Tetrahymena pryiformis strain W was purified about 900-fold by (NH4)2SO4 precipitation, gel filtration and ion exchange chromatography. The purified acid phosphatase was homogenous as judged by polycrylamide gel electrophoresis and was found to be a glycoprotein. Its carbohydrate content was about 10% of the total protein content. The native enzyme has a molecular weight of 120 000 as determined by gel filtration and 61 000 as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The acid phosphatase thus appears to consist of two subunits of equal size. The amino acid analysis revealed a relatively high content of asparic acid, glutamic acid and leucine. The purified acid phosphatase from Tetrahymena had a rather broad substrate specificity; it hydrolyzed organic phosphates, nucleotide phosphates and hexose phosphates, but had no diesterase activity. The Km values determined with p-nitrophenyl phosphate, adenosine 5′-phosphate and glucose 6-phosphate were 3.1·10?4 M, 3.9·10?4 M and 1.6·10?3 M, respectively. The optima pH for hydrolysis of three substrates were similar (pH 4.6). Hg2+ and Fe3+ at 5 mM were inhibitory for the purified acid phosphatase, and fluoride, L-(+)-tartaric acid and molybdate also inhibited its cavity at low concentrations. The enzyme was competitively inhibited by NaF (Ki=5.6·10?4 M) and by L-(+)-tartaric acid (Ki = 8.5·10?5 M), while it was inhibited noncompetitively by molybdate Ki = 5.0·10?6 M). The extracellular acid phosphatase purified from Tetrahymena was indistinguishable from the intracellular enzyme in optimum pH, Km, thermal stability and inhibition by NaF. 相似文献
16.
The advantage of using DNA content as a biochemical parameter because the results it gives are directly related to cellularity is discussed. As examples, comparisons of acid phosphatase and cathepsin D activities in rat liver and hepatoma and of acid phosphatase in human normal breast tissue and adenocarcinoma are considered. Contradictory results are obtained, depending whether they are related to DNA content, fresh tissue weight, or protein content. 相似文献
17.
Activity of acid phosphatase secreted by mycelia ofPholiota nameko on cultivation for 30d in Pi-depleted medium was 88-fold higher than the corresponding activity in the Pi-supplied medium.
One isozyme of the secreted acid phosphatases was purified from the culture filtrate of Pi-depleted medium by ammonium sulfate
fractionation and cation exchange chromatography. The purified enzyme was homogeneous on electrophoresis. Gel filtration analysis
showed change chromatography. The purified enzyme was homogeneous on electrophoresis. Gel filtration analysis showed that
the native molecule had a molecular weight of 117,000. The molecular weight on gel electrophoresis with SDS was 52,000, indicating
that the native form of the enzyme was a homodimer. The optimum pH and temperature of the enzyme were, 5.5 and 45°C, respectively,
and the isoelectric point of the enzyme was pH 6.9. Adsorption on Con A-Sepharose and periodic-Schiff stain suggested that
the enzyme is a glycoprotein. The enzyme hydrolyzed a wide variety of phosphate esters, nucleoside phosphates, sugar phosphates,
and phosphorylated amino acids. Cu2+, Fe2+, Hg2+, iodoacetate, molybdate, tartaric acid, and SDS inhibited the enzyme activity. Fe3+ (1 mM), Triton X-100, methanol, and ethanol activated it. Fifteen residues of the N-terminal amino acid sequence were determined. 相似文献
18.
Protein tyrosine phosphatase sigma (PTPσ) plays a vital role in neural development. The extracellular domain of PTPσ binds to various proteoglycans, which control the activity of 2 intracellular PTP domains (D1 and D2). To understand the regulatory mechanism of PTPσ, we carried out structural and biochemical analyses of PTPσ D1D2. In the crystal structure analysis of a mutant form of D1D2 of PTPσ, we unexpectedly found that the catalytic cysteine of D1 is oxidized to cysteine sulfenic acid, while that of D2 remained in its reduced form, suggesting that D1 is more sensitive to oxidation than D2. This finding contrasts previous observations on PTPα. The cysteine sulfenic acid of D1 was further confirmed by immunoblot and mass spectrometric analyses. The stabilization of the cysteine sulfenic acid in the active site of PTP suggests that the formation of cysteine sulfenic acid may function as a stable intermediate during the redox-regulation of PTPs. 相似文献
19.
Antibodies were raised against one cytoplasmic and two membrane-bound acid phosphatases purified from yam tubers (Dioscorea cayenensis rotundata). Experiments of immunoinactivation and immunoelectrophoresis revealed cross-immunological reactions between the cytoplasmic enzyme (acid phosphatase A) and one of two membrane-bound counterparts (acid phosphatase B) suggesting that these molecules share common antigenic determinants. The antibodies raised against the other membrane-bound enzyme (acid phosphatase C) only inhibited and precipitated this enzyme. 相似文献
20.
Sin-Lui Yeung Chiwai Cheng Thomas K.O. Lui Jimmy S.H. Tsang Wing-Tat Chan Boon L. Lim 《Gene》2009,440(1-2):1-8
Purple acid phosphatases (PAP) are a group of dimetallic phosphohydrolase first identified in eukaryotes. Bioinformatics analysis revealed 57 prokaryotic PAP-like sequences in the genomes of 43 bacteria and 4 cyanobacteria species. A putative PAP gene (BcPAP) from the bacteria Burkholderia cenocepacia J2315 was chosen for further studies. Synteny analysis showed that this gene is present as an independent gene in most of the members of the genus Burkholderia. The predicted 561 a.a. polypeptide of BcPAP was found to harbour all the conserved motifs of the eukaryotic PAPs and an N-terminal twin-arginine translocation signal. Expression and biochemical characterization of BcPAP in Escherichia coli revealed that this enzyme has a relatively narrow substrate spectrum, preferably towards phosphotyrosine, phosphoserine and phosphoenolpyruvate. Interestingly, this enzyme was found to have a pH optimum at 8.5, rather than an acidic optima exhibited by eukaryotic PAPs. BcPAP contains a dimetallic ion centre composed of Fe and Zn, and site-directed mutagenesis confirmed that BcPAP utilizes the invariant residues for metal-ligation and catalysis. The enzyme is secreted by the wild type bacteria and its expression is regulated by the availability of orthophosphate. Our findings suggest that not all members in the PAP family have acidic pH optimum and broad substrate specificity. 相似文献