Expression and characterization of recombinant thermostable alkaline phosphatase from a novel thermophilic bacterium Thermus thermophilus XM

A gene （tap） encoding a thermostable alkaline phosphatase from the thermophilic bacterium Thermus thermophilus XM was cloned and sequenced. It is 1506 bp long and encodes a protein of 501 amino acid residues with a calculated molecular mass of 54.7 kDa. Comparison of the deduced amino acid sequence with other alkaline phosphatases showed that the regions in the vicinity of the phosphorylation site and metal binding sites are highly conserved. The recombinant thermostable alkaline phosphatase was expressed as a His6-tagged fusion protein in Escherichia coli and its enzymatic properties were characterized after purification. The pH and temperature optima for the recombinant thermostable alkaline phosphatases activity were pH 12 and 75 ℃. As expected, the enzyme displayed high thermostability, retaining more than 50% activity after incubating for 6 h at 80 ℃. Its catalytic function was accelerated in the presence of 0.1 mM Co^2＋, Fe^2＋, Mg^2＋, or Mn^2＋ but was strongly inhibited by 2.0 mM Fe^2＋. Under optimal conditions, the Michaelis constant （Kin） for cleavage of p-nitrophenyl-phosphate was 0.034 mM. Although it has much in common with other alkaline phosphatases, the recombinant thermostable alkaline phosphatase possesses some unique features, such as high optimal pH and good thermostability.     

Bacillus subtilis alkaline phosphatases III and IV. Cloning, sequencing, and comparisons of deduced amino acid sequence with Escherichia coli alkaline phosphatase three-dimensional structure

Bacillus subtilis has an alkaline phosphatase multigene family. Two members of this gene family, phoAIII and phoAIV, were cloned, taking advantage of in vitro constructed strains containing a plasmid insertion within one or the other of the structural genes. The DNA sequences of the two genes showed approximately 64% identity at the DNA level and 63% identity in the deduced primary amino acid sequences. The phoAIII and phoAIV genes code for predicted proteins of 47,149 and 45,935 Da, respectively. Comparison of the deduced primary amino acid sequence of the mature proteins with other sequenced alkaline phosphatases from Escherichia coli, yeast, and humans shows 25-30% identity. Based on the refined crystal structure of E. coli alkaline phosphatase, it appears that the active site and the core of the structure are retained in both Bacillus alkaline phosphatases. However, both proteins are truncated at the amino terminus compared with other mature alkaline phosphatases, three sizable surface loops of E. coli are deleted, and a minidomain is replaced with a larger domain in the model. Neither Bacillus alkaline phosphatase sequenced contains any cysteine residues, an amino acid implicated in intrachain disulfide bond formation in other alkaline phosphatases.     

Thermolabile alkaline phosphatase from Northern shrimp (Pandalus borealis): protein and cDNA sequence analyses

Sequence analysis of short fragments resulting from trypsin digestion of the thermolabile shrimp alkaline phosphatase (SAP) from Northern shrimp Pandalus borealis formed the basis for amplification of its encoding cDNA. The predicted protein sequence was recognized as containing the consensus alkaline phosphatase motif comprising the active site of this protein family. Protein sequence homology searches identified several eukaryote alkaline phosphatases with which the 475-amino acid SAP polypeptide revealed shares 45% amino acid sequence identity. Residues for potential metal binding seem to be conserved in these proteins. The predicted 54-kDa molecular mass of SAP is smaller than previously reported, but is consistent with our recent SDS-PAGE analysis of the native protein. Compared to its homologs, the shrimp enzyme has a surplus of negatively charged amino acids, while the relative number of prolines is lower and the frequency of aromatic residues is higher than in mesophilic counterparts.     

Cloning of cold-active alkaline phosphatase gene of a psychrophile,Shewanella sp., and expression of the recombinant enzyme

A psychrophilic alkaline phosphatase (EC 3.1.3.1) from Shewanella sp. is a cold-active enzyme that has high catalytic activity at low temperature [Ishida et al. (1998) Biosci. Biotechnol. Biochem., 62, 2246-2250]. Here, we identified the nucleotide sequence of a gene encoding the enzyme after cloning with the polymerase chain reaction (PCR) and inverted PCR techniques. The deduced amino acid sequence of the enzyme contained conserved amino acids found among mesophilic alkaline phosphatases and showed some structural characteristics including a high content of hydrophobic amino acid residues and the lack of single alpha-helix compared with the alkaline phosphatase of Escherichia coli, which were possibly efficient for catalytic reaction at low temperatures. The recombinant enzyme expressed in E. coli was purified to homogeneity with the molecular mass of 41 kDa. The recombinant enzyme had a specific activity of 1,500 units/mg and had high catalytic activity at low temperatures.     

Cloning of an alkaline phosphatase gene from the moderately thermophilic bacterium<Emphasis Type="Italic"> Meiothermus ruber</Emphasis> and characterization of the recombinant enzyme

A gene that codes for an alkaline phosphatase was cloned from the thermophilic bacterium Meiothermus ruber, and its nucleotide sequence was determined. The deduced amino acid sequence indicates that the enzyme precursor including the putative signal sequence is composed of 503 amino acid residues and has an estimated molecular mass of 54,229 Da. Comparison of the peptide sequence with that of the prototype alkaline phosphatase from Escherichia coli revealed conservation of the regions in the vicinity of the corresponding phosphorylation site and metal binding sites. The protein was expressed in E. coli and its enzymatic properties were characterized. In the absence of exogenously added metal ions, activity was negligible; to obtain maximal activity, addition of free Mg²⁺ ions was required. Zn²⁺ ions had an inhibitory effect on the activity of the M. ruber enzyme. The pH and temperature optima for activity were found to be 11.0 and 62°C, respectively. The enzyme was moderately thermostable: it retained about 50% activity after incubation for 6 h at 60°C, whereas at 80°C it was completely inactivated within 2 h. The Michaelis constant for cleavage of 4-nitrophenylphosphate was 0.055 mM. While having much in common with other alkaline phosphatases, the M. ruber enzyme presents some unique features, such as a very narrow pH range for activity and an absolute requirement for magnesium for activity.Communicated by G. P. Georgiev     

Gene cloning, overproduction, and characterization of thermolabile alkaline phosphatase from a psychrotrophic bacterium

The gene encoding alkaline phosphatase from the psychrotrophic bacterium Shewanella sp. SIB1 was cloned, sequenced, and overexpressed in Escherichia coli. The recombinant protein was purified and its enzymatic properties were compared with those of E. coli alkaline phosphatase (APase), which shows an amino acid sequence identity of 37%. The optimum temperature of SIB1 APase was 50 degrees C, lower than that of E. coli APase by 30 degrees C. The specific activity of SIB1 APase at 50 degrees C was 3.1 fold higher than that of E. coli APase at 80 degrees C. SIB1 APase lost activity with a half-life of 3.9 min at 70 degrees C, whereas E. coli APase lost activity with a half-life of >6 h even at 80 degrees C. Thus SIB1 APase is well adapted to low temperatures. Comparison of the amino acid sequences of SIB1 and E. coli APases suggests that decreases in electrostatic interactions and number of disulfide bonds are responsible for the cold-adaptation of SIB1 APase.     

Bacillus licheniformis MC14 alkaline phosphatase I gene with an extended COOH-terminus

Bacterial alkaline phosphatases (APases), except those isolated from Bacillus licheniformis, are approximately 45-kDa proteins while eucaryotic alkaline phosphatases are 60 kDa. To answer the question of whether the apparent 60-kDa alkaline phosphatase from Bacillus licheniformis accurately reflected the size of the protein, the entire gene was analyzed. DNA sequence analysis of the alkaline phosphatase I (APaseI) gene of B. licheniformis MC14 indicated that the gene could code for a 60-kDa protein of 553 amino acids. The deduced protein sequence of APaseI showed about 32% identity to those of B. subtilis APase III and IV and had apparent sequence homologies in the core structure and active sites that are conserved among APases of various sources. The extra carboxy-terminal sequence of APaseI, which made the enzyme bigger than other procaryotic APases, was not homologous to those of eucaryotic APases. The amino acid composition of APaseI was most similar to that of salt-dependent APase among the isozymes of B. licheniformis MC14. Another open reading frame of 261 amino acids was present 142 nucleotide upstream of the APaseI gene and its predicted amino acid sequence showed 68% identity to that of glucose dehydrogenase of B. megaterium.     

Altering of the metal specificity of Escherichia coli alkaline phosphatase

Analysis of sequence alignments of alkaline phosphatases revealed a correlation between metal specificity and certain amino acid side chains in the active site that are metal-binding ligands. The Zn(2+)-requiring Escherichia coli alkaline phosphatase has an Asp at position 153 and a Lys at position 328. Co(2+)-requiring alkaline phosphatases from Thermotoga maritima and Bacillus subtilis have a His and a Trp at these positions, respectively. The mutations D153H, K328W, and D153H/K328W were induced in E. coli alkaline phosphatase to determine whether these residues dictate the metal dependence of the enzyme. The wild-type and D153H enzymes showed very little activity in the presence of Co(2+), but the K328W and especially the D153H/K328W enzymes effectively use Co(2+) for catalysis. Isothermal titration calorimetry experiments showed that in all cases except for the D153H/K328W enzyme, a possible conformation change occurs upon binding Co(2+). These data together indicate that the active site of the D153H/K328W enzyme has been altered significantly enough to allow the enzyme to utilize Co(2+) for catalysis. These studies suggest that the active site residues His and Trp at the E. coli enzyme positions 153 and 328, respectively, at least partially dictate the metal specificity of alkaline phosphatase.     

Mutagenesis of conserved residues within the active site of Escherichia coli alkaline phosphatase yields enzymes with increased kcat.

The likelihood for improvement in the catalytic properties of Escherichia coli alkaline phosphatase was examined using site-directed mutagenesis. Mutants were constructed by introducing sequence changes into nine preselected amino acid sites within 10 A of the catalytic residue serine 102. When highly conserved residues in the family of alkaline phosphatases were mutated, many of the resulting enzymes not only maintained activity, but also exhibited greatly improved kcat. Of approximately 170 mutant enzymes screened, 5% (eight mutants) exhibited significant increases in specific activity. In particular, a substitution by serine of a totally invariant Asp101 resulted in a 35-fold increase of specific activity over wild-type at pH 10.0. Up to 6-fold increases of the kcat/Km ratio were observed.     

The active-site and amino-terminal amino acid sequence of bovine intestinal alkaline phosphatase

The active site of bovine intestinal alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) was labeled with [32P]Pi, a radioactive CNBr peptide was isolated and the amino acid sequence was determined. The sequence of the active-site peptide has limited homology (26%) with the active-site sequence of Escherichia coli alkaline phosphatase except for the ten residues immediately flanking the active-site serine (70%). A possible amino acid sequence deduced from the amino acid composition of an active-site tryptic peptide from human placental alkaline phosphatase is very similar to the bovine intestinal active-site sequence. The amino-terminal sequence of bovine intestinal alkaline phosphatase is homologous (69%) with the human placental enzyme but not with the E. coli phosphatase.     

A method for enucleation of Saccharomyces cerevisiae

Abstract We have analysed the response of the acidophilic chemolithotroph Thiobacillus ferrooxidans to phosphate starvation. Cultivation of the bacteria in the absence of added phosphate induced a remarkable filamentation of the cells. Polyacrylamide gel electrophoresis revealed several proteins whose levels increased upon phosphate limitation, as well as some polypeptides that were exclusively synthesized under this growth limitation. One of the proteins whose level increased by the lack of phosphate was apparently an acid phosphatase with a pH optimum of about 3.8, and a molecular mass of 26 kDa, which was located in the periplasm. The N-terminal sequence of a 26 kDa protein derepressed by starvation, which may correspond to the T. ferrooxidans phosphatase, showed 30% and 35% identity with the known sequence of Lysobacter enzymogenes and Escherichia coli alkaline phosphatases, respectively.     

Identification of the adipocyte acid phosphatase as a PAO-sensitive tyrosyl phosphatase.

We have partially purified an 18-kDa cytoplasmic protein from 3T3-L1 cells, which dephosphorylates pNPP and the phosphorylated adipocyte lipid binding protein (ALBP), and have identified it by virtue of kinetic and immunological criteria as an acid phosphatase (EC 3.1.3.2). The cytoplasmic acid phosphatase was inactivated by phenylarsine oxide (PAO) (Kinact = 10 microM), and the inactivation could be reversed by the dithiol, 2,3-dimercaptopropanol (Kreact = 23 microM), but not the monothiol, 2-mercaptoethanol. Cloning of the human adipocyte acid phosphatase revealed that two isoforms exist, termed HAAP alpha and HAAP beta (human adipocyte acid phosphatase), which are distinguished by a 34-amino acid isoform-specific domain. Sequence analysis shows HAAP alpha and HAAP beta share 74% and 90% identity with the bovine liver acid phosphatase, respectively, and 99% identity with both isoenzymes of the human red cell acid phosphatase but no sequence similarity to the protein tyrosine phosphatases (EC 3.1.3.48). HAAP beta has been cloned into Escherichia coli, expressed, and purified as a glutathione S-transferase fusion protein. Recombinant HAAP beta was shown to dephosphorylate pNPP and phosphoALBP and to be inactivated by PAO and inhibited by vanadate (Ki = 17 microM). These results describe the adipocyte acid phosphatase as a cytoplasmic enzyme containing conformationally vicinal cysteine residues with properties that suggest it may dephosphorylate tyrosyl phosphorylated cellular proteins.     

Plasmodium falciparum protein phosphatase type 1 functionally complements a glc7 mutant in Saccharomyces cerevisiae

We have identified a new homologue of protein phosphatase type 1 from Plasmodium falciparum, designated PfPP1, which shows 83-87% sequence identity with yeast and mammalian PP1s at the amino acid level. The PfPP1 sequence is strikingly different from all other P. falciparum Ser/Thr phosphatases cloned so far. The deduced 304 amino acid sequence revealed the signature sequence of Ser/Thr phosphatase LRGNHE, and two putative protein kinase C and five putative casein kinase II phosphorylation sites. Calyculin A, a potent inhibitor of Ser/Thr phosphatase 1 and 2A showed hyperphosphorylation of a 51kDa protein among other parasite proteins. Okadaic acid on the other hand, was without any effect suggesting that PP1 activity might predominate over PP2A activity in intra-erythrocytic P. falciparum. Complementation studies showed that PfPP1 could rescue low glycogen phenotype of Saccharomyces cerevisiae glc7 (PP1) mutant, strongly suggesting functional interaction of PfPP1 and yeast proteins involved in glycogen metabolism.     

Isolation of a mRNA that encodes a putative intestinal alkaline phosphatase regulated by 1,25-dihydroxyvitamin D-3

Using differential hybridization techniques to screen a rat intestinal cDNA library we isolated a cDNA whose predicted amino acid sequence exhibits a high degree of homology to the alkaline phosphatases. The predicted cDNA sequence has 79% identity at the amino acid level to the rat intestinal alkaline phosphatase, and shows approx. 70% homology to other human and rat alkaline phosphatases. The corresponding mRNA is markedly increased by 6 h after a single dose of 1,25-dihydroxyvitamin D-3. The mRNA is also increased by 24-homologated analogs of 1,25-dihydroxyvitamin D-3 that do not increase calcium transport.     

Alkaline phosphatase from the hyperthermophilic bacterium T. maritima requires cobalt for activity

The hyperthermophilic bacterium Thermotoga maritima encodes a gene sharing sequence similarities with several known genes for alkaline phosphatase (AP). The putative gene was isolated and the corresponding protein expressed in Escherichia coli, with and without a predicted signal sequence. The recombinant protein showed phosphatase activity toward the substrate p-nitrophenyl-phosphate with a k(cat) of 16 s(-1) and a K(m) of 175 microM at a pH optimum of 8.0 when assayed at 25 degrees C. T. maritima phosphatase activity increased at high temperatures, reaching a maximum k(cat) of 100 s(-1), with a K(m) of 93 microM at 65 degrees C. Activity was stable at 65 degrees C for >24 h and at 90 degrees C for 5 h. Phosphatase activity was dependent on divalent metal ions, specifically Co(II) and Mg(II). Circular dichroism spectra showed that the enzyme gains secondary structure on addition of these metals. Zinc, the most common divalent metal ion required for activity in known APs, was shown to inhibit the T. maritima phosphatase enzyme at concentrations above 0.3 moles Zn: 1 mole monomer. All activity was abolished in the presence of 0.1 mM EDTA. The T. maritima AP primary sequence is 28% identical when compared with E. coli AP. Based on a structural model, the active sites are superimposable except for two residues near the E. coli AP Mg binding site, D153 and K328 (E. coli numbering) corresponding to histidine and tryptophan in T. maritima AP, respectively. Sucrose-density gradient sedimentation experiments showed that the protein exists in several quaternary forms predominated by an octamer.     

Directed evolution of the 5'-untranslated region of the phoA gene in Escherichia coli simultaneously yields a stronger promoter and a stronger Shine-Dalgarno sequence

Directed evolution has been widely applied for gene improvement through random mutagenesis of coding sequences. Through error-prone PCR both in the coding sequence and the regulatory sequence of E. coli alkaline phosphatase, the cellular enzyme activity has been efficiently enhanced. Sequence analysis revealed that the resultant mutant 34-B12, which showed a sevenfold increased enzyme activity at the cellular level, contains three mutations in the regulatory sequence and another three mutations in the coding sequence. Activity assays of the enzyme containing the corresponding amino acid substitutions proved that the amino acid mutations contribute only to a small portion to the increased cellular enzyme activity. So the mutations in the 5'-untranslated region were analyzed separately and combinationally. The results suggested that one mutation yielded a stronger promoter and the other two mutations both elevated the E. coli alkaline phosphatase expression at the translational level; moreover, a stronger Shine-Dalgarno sequence was generated.     

Calix[4]arene methylenebisphosphonic acids: the features of alkaline phosphatases inhibition

The inhibition of alkaline phosphatases by calix[4]arenes functionalysed at the macrocyclic upper rim by one or two methylenebisphosphonic acid fragments has been investigated. It is established, that calix[4]arene bismethylenebisphosphonic acid displayed stronger inhibition of alkaline phosphatase from bovine intestine mucosa than calix[4]arene methylenebisphosphonic acid. At the same time, the both inhibitors showed almost similar levels of inhibitory activities in respect of bovine kidney alkaline phosphatase or E. coli alkaline phosphatase. The tested compounds were docked computationally to the active site of the E. coli alkaline phosphatase. On the basis of results obtained the possible binding modes of inhibitors were analysed.     

3-D Model of the bee venom acid phosphatase: Insights into allergenicity

The acid phosphatase Api m 3 is the major allergen of the honeybee venom. Except for the amino acid sequence, no other structural information for the enzyme is available. We applied homology modeling to assign the three-dimensional structure of Api m 3. The structure of the homodimeric human prostatic acid phosphatase was used to model the Api m 3 tertiary structure. IgE epitopes and antigenic sites were predicted using programs based on the structure of known epitopes and analysis of the 3-D model. The model of Api m 3 revealed an active site similar to those of the histidine—type acid phosphatases with conservation of the catalytically important residues. The observed substitutions in the phosphate ion binding site suggest differences in the substrate specificity in comparison to other acid phosphatases. The analysis of the Api m 3 three-dimensional model revealed a very likely mechanism of enzyme action.