Developing robust protein analysis profiles to identify bacterial acid phosphatases in genomes and metagenomic libraries.

Phylogenetic analysis of more than 4000 annotated bacterial acid phosphatases was carried out. Our analysis enabled us to sort these enzymes into the following three types: (1) class B acid phosphatases, which were distantly related to the other types, (2) class C acid phosphatases and (3) generic acid phosphatases (GAP). Although class B phosphatases are found in a limited number of bacterial families, which include known pathogens, class C acid phosphatases and GAP proteins are found in a variety of microbes that inhabit soil, fresh water and marine environments. As part of our analysis, we developed three profiles, named Pfr-B-Phos, Pfr-C-Phos and Pfr-GAP, to describe the three groups of acid phosphatases. These sequence-based profiles were then used to scan genomes and metagenomes to identify a large number of formerly unknown acid phosphatases. A number of proteins in databases annotated as hypothetical proteins were also identified by these profiles as putative acid phosphatases. To validate these in silico results, we cloned genes encoding candidate acid phosphatases from genomic DNA or recovered from metagenomic libraries or genes synthesized in vitro based on protein sequences recovered from metagenomic data. Expression of a number of these genes, followed by enzymatic analysis of the proteins, further confirmed that sequence similarity searches using our profiles could successfully identify previously unknown acid phosphatases.

Identification of Tea Plant Purple Acid Phosphatase Genes and Their Expression Responses to Excess Iron.

Purple acid phosphatase (PAP) encoding genes are a multigene family. PAPs require iron (Fe) to exert their functions that are involved in diverse biological roles including Fe homeostasis. However, the possible roles of PAPs in response to excess Fe remain unknown. In this study, we attempted to understand the regulation of PAPs by excess Fe in tea plant (Camellia sinensis). A genome-wide investigation of PAP encoding genes identified 19 CsPAP members based on the conserved motifs. The phylogenetic analysis showed that PAPs could be clustered into four groups, of which group II contained two specific cysteine-containing motifs "GGECGV" and "YERTC". To explore the expression patterns of CsPAP genes in response to excessive Fe supply, RNA-sequencing (RNA-seq) analyses were performed to compare their transcript abundances between tea plants that are grown under normal and high iron conditions, respectively. 17 members were shown to be transcribed in both roots and leaves. When supplied with a high amount of iron, the expression levels of four genes were significantly changed. Of which, CsPAP15a, CsPAP23 and CsPAP27c were shown as downregulated, while the highly expressed CsPAP10a was upregulated. Moreover, CsPAP23 was found to be alternatively spliced, suggesting its post-transcriptional regulation. The present work implicates that some CsPAP genes could be associated with the responses of tea plants to the iron regime, which may offer a new direction towards a further understanding of iron homeostasis and provide the potential approaches for crop improvement in terms of iron biofortification.

A novel immunoassay for the determination of tartrate-resistant acid phosphatase 5b from rat serum.

Osteoclasts secrete tartrate-resistant acid phosphatase 5b (TRACP 5b) into the circulation. We have developed an immunoassay for the determination of rat TRACP 5b activity. Intra-assay variation of the immunoassay was 4.5%, interassay variation was 3.8%, dilution linearity was 104.6 +/- 7.6%, and recovery of recombinant rat TRACP was 99.1 +/- 5.8%. We studied serum TRACP 5b as a marker of bone resorption using orchidectomized (ORC) rats as a model for osteoporosis and age-matched sham-operated rats as controls in a 6-month study. After the operation, trabecular bone mineral density decreased significantly more in the ORC group than in the sham group, whereas cortical bone mineral density increased similarly in both groups. Serum TRACP 5b activity was significantly elevated within the first week after ORC, returned to the control level in the third week, and was not increased above the sham level at any of the later time points. At 6 months, trabecular bone volume was 80% lower in ORC rats than in controls. Osteoclast number per trabecular bone perimeter was slightly increased, but the absolute number of osteoclasts in trabecular bone was significantly decreased. These results suggest that absolute bone resorption is increased within the first week after ORC. Later, it is decreased because there is less bone to be resorbed. However, relative bone resorption (compared with the amount of remaining bone) is still increased, leading to further bone loss. We conclude that serum TRACP 5b is a useful marker for monitoring changes in the bone resorption rate in rat ORC model.

Isolation and characterization of the genes encoding mouse and human type-5 acid phosphatase.

The gene (mT5AP) encoding murine type-5 acid phosphatase has been isolated and completely sequenced while the gene (hT5AP) encoding human T5AP has been partly sequenced. The murine gene spans 4 kb and contains five exons. Exon 1 is completely non-coding and exon 2 starts with the initiation codon in both mT5AP and hT5AP. The positions of the intron/exon boundaries are completely conserved between mT5AP and hT5AP, but are distinct from the gene encoding the related porcine protein, uteroferrin (Utf). There is strong homology at both the nucleotide (nt) and amino acid (aa) levels between the inferred mouse cDNA and the sequences of rat T5AP and hT5AP, and pig Utf. The mT5AP and hT5AP genes were found to have multiple transcription start points (tsp) by primer extension analysis, consistent with the absence of a consensus TATA box. The sequences for the 5'-flanking regions of mT5AP and hT5AP were determined to -1.6 and -1.0 kb, respectively, relative to the tsp. A 2-kb segment of the mT5AP 5' flanking region linked to a luciferase-encoding reporter gene (Luc) was sufficient to direct tissue-specific transcription in the mouse macrophage cell line, RAW264. Significant sequence similarity between the mT5AP and hT5AP promoters is restricted to the most proximal 200 bp, which also resembles the porcine Utf gene, and a 300-bp segment 700 bp upstream. A progesterone-response element is present only in the mouse promoter and the estrogen- and iron-response elements described previously in the pig gene are absent from both the mouse and human genes. These differences may result in distinctive regulation of T5AP and Utf expression.

The Aspergillus niger (ficuum) aphA gene encodes a pH 6.0-optimum acid phosphatase.

We have used the Aspergillus niger (An) aphA gene as a probe and cloned the A. ficuum (Af) SRRC 265 gene encoding an extracellular pH 6.0-optimum acid phosphatase (APase6) from a genomic library. The identity of the Af aphA gene was confirmed and its nucleotide (nt) sequence verified by comparing its deduced amino acid (aa) sequence to that of purified Af APase6. A comparison of the nt sequences of the An and Af genes suggested that errors were made in the previously reported An aphA sequence. Several regions of the An aphA were resequenced and the mistakes corrected. With its nt sequence corrected, the An aphA is nearly identical to the cloned Af gene encoding APase6, and in 90.4% agreement in the coding regions. Both genes have three conserved introns and when translated, both nt sequences code for a polypeptide of 614 aa. There is now evidence that the two cloned genes are homologous and code for acid phosphatases that are 96% identical.

Molecular properties of multiple forms of acid phosphatase from horse liver.

1. Horse liver acid phosphatase was separated into two partially purified fractions differing in molecular weight (enzyme I about 100 00, enzyme II about 25 000). 2. Enzyme I was separated into several subfractions by DEAE-cellulose chromatography and isoelectric focusing. 3. Molecular weight, sedimentation coefficient and effective molecular radii were determined for acid phosphatases I and II by gel filtration and density-gradient centrifugation.

Characterization of a phosphatase secreted by Staphylococcus aureus strain 154, a new member of the bacterial class C family of nonspecific acid phosphatases.

An acid phosphatase, designated SapS, hydrolyzing p-nitrophenyl phosphate (pNPP), was identified and characterized from the culture supernatant of a Staphylococcus aureus strain isolated from vegetables. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the protein indicated an estimated molecular mass of 30 kDa. The enzyme displayed optimum activity at 40 degrees C and pH 5. Characterization of the phosphatase in a reconstitution assay showed that MgCl2 and Triton X-100, respectively, restored maximal activity, but not CaCl2 The phosphatase activity was affected by EDTA and sodium molybdate. The DNA sequence encoding SapS was cloned and sequenced. The putative acid phosphatase gene encodes a protein of 296 amino acids with a 31-residue signal peptide. Database searches revealed significant structural homology of SapS to several proteins belonging to the bacterial class C family of nonspecific acid phosphatases. Comparison of the sequences indicated that despite a low level of overall conservation between the proteins, four conserved sequence motifs could be identified.

Comparative analysis of PvPAP gene family and their functions in response to phosphorus deficiency in common bean.

BACKGROUND: Purple acid phosphatases (PAPs) play a vital role in adaptive strategies of plants to phosphorus (P) deficiency. However, their functions in relation to P efficiency are fragmentary in common bean. PRINCIPAL FINDINGS: Five PvPAPs were isolated and sequenced in common bean. Phylogenetic analysis showed that PvPAPs could be classified into two groups, including a small group with low molecular mass, and a large group with high molecular mass. Among them, PvPAP3, PvPAP4 and PvPAP5 belong to the small group, while the other two belong to the large group. Transient expression of 35S:PvPAPs-GFP on onion epidermal cells verified the variations of subcellular localization among PvPAPs, suggesting functional diversities of PvPAPs in common bean. Quantitative PCR results showed that most PvPAPs were up-regulated by phosphate (Pi) starvation. Among them, the expression of the small group PvPAPs responded more to Pi starvation, especially in the roots of G19833, the P-efficient genotype. However, only overexpressing PvPAP1 and PvPAP3 could result in significantly increased utilization of extracellular dNTPs in the transgenic bean hairy roots. Furthermore, overexpressing PvPAP3 in Arabidopsis enhanced both plant growth and total P content when dNTPs were supplied as the sole external P source. CONCLUSIONS: The results suggest that PvPAPs in bean varied in protein structure, response to P deficiency and subcellular localization. Among them, both PvPAP1 and PvPAP3 might function as utilization of extracellular dNTPs.

Purple acid phosphatase-like sequences in prokaryotic genomes and the characterization of an atypical purple alkaline phosphatase from Burkholderia cenocepacia J2315.

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.

Bacterial nonspecific acid phosphohydrolases: physiology, evolution and use as tools in microbial biotechnology.

Bacterial nonspecific acid phosphohydrolases (NSAPs) are secreted enzymes, produced as soluble periplasmic proteins or as membrane-bound lipoproteins, that are usually able to dephosphorylate a broad array of structurally unrelated substrates and exhibit optimal catalytic activity at acidic to neutral pH values. Bacterial NSAPs are monomeric or oligomeric proteins containing polypeptide components with an M(r) of 25-30 kDa. On the basis of amino acid sequence relatedness, three different molecular families of NSAPs can be distinguished, indicated as molecular class A, B and C, respectively. Members of each class share some common biophysical and functional features, but may also exhibit functional differences. NSAPs have been detected in several microbial taxa, and enzymes of different classes can be produced by the same bacterial species. Structural and phyletic relationships exist among the various bacterial NSAPs and some other bacterial and eucaryotic phosphohydrolases. Current knowledge on bacterial NSAPs is reviewed, together with analytical tools that may be useful for their characterization. An overview is also presented concerning the use of bacterial NSAPs in biotechnology.

Acid phosphatase activity in human semen.

Acid phosphatase (ACP) was studied in normal and abnormal human semen of individuals not suffering from testosterone deficiency. Study of ACP activity was performed on 365 men referred to the OPD clinic of L.T.M.G. Hospital, Bombay. Semen samples were classified into five groups according to sperm concentration, vitality, motility, and morphology. An inverse correlation was found between enzyme activity and sperm concentration. Statistical analysis revealed that ACP activity was maximal in the azoospermic group, and decreases as the sperm concentration increases. On the basis of this, measurement of ACP in human semen may be useful in clinical practice.

An enzyme with a double identity: purple acid phosphatase and tartrate-resistant acid phosphatase.

The tartrate-resistant acid phosphatases or purple acid phosphatases constitute a class of related mammalian enzymes. Spectroscopic and magnetic studies have revealed that the purple phosphatases contain a novel dinuclear iron active site that is responsible for the purple color. More biologically and biomedically oriented research has shown that the tartrate-resistant acid phosphatases generally occur in osteoclasts and white blood cells, where they appear to be localized in lysosomes or similar organelles. Despite the different names given the enzymes by researchers in the two fields, recent sequence determinations and immunological studies indicate that the enzymes are identical. The status of research in both fields is reviewed in an attempt to present a unified picture of the structure, function, and mode of action of these unique metalloproteins.

Characterization of high- and low-molecular weight zinc-dependent acid phosphatases in bovine liver.

We have purified two forms of Zn2+-dependent acid phosphatase (Zn2+-APase) from bovine liver, both of which require Zn2+ to hydrolyze the substrate p-nitrophenyl phosphate in an acidic environment. The apparent molecular weights of these two forms of Zn2+-APase were estimated to be about 100,000 and 62,000 by gel filtration, and about 44,000 and 31,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, respectively. The low-molecular weight (LMW) Zn2+-APase catalyzed the hydrolysis of myo-inositol-1-phosphate in the presence of 3 mM Mg2+ at physiological pH, but the high-molecular weight (HMW) enzyme did not. The LMW-Zn2+-APase of bovine liver was recognized by polyclonal antibodies developed against the Zn2+-APase of bovine brain, but the HMW-Zn2+-APase was not.

Degradation of AMP in erythrocytes of man. Evidence for a cytosolic phosphatase activity.

By means of selective inhibitors of adenosine deaminase and adenosine kinase, the contributions of two competing pathways for the breakdown of adenosine nucleotides in erythrocytes of man were examined. Under nearly physiological conditions in vitro the main pathway for the irreversible breakdown proceeds from AMP via IMP and inosine to hypoxanthine. Its rate amounts to 12 mumol AMP/l cells X h. At the same time about three times as much AMP, about 40 mumol/l cells X h, are degraded by way of dephosphorylation to adenosine. However, this pathway does not contribute significantly to the production of hypoxanthine, since the adenosine formed is rephosphorylated by adenosine kinase. Both AMP and IMP are dephosphorylated by an unspecific cytosolic acid phosphatase, the maximal activity of which amounts to 660 mumol nucleotide/l cells X h.

The lower molecular weight acid phosphatase from the frog liver: isolation of homogeneous AcPase III and IV representing glycoforms with different bioactivity.

1. AcPase III and AcPase IV, the major enzyme forms of the LMW AcPase of the frog (Rana esculenta) liver were resolved and purified to homogeneity. 2. AcPase III and IV showed a single protein band on SDS-PAGE corresponding to a mol. wt (Mr) of about 35,000. The Mr of the native enzyme forms were 33,200 (gel electrophoresis) and 38,200 +/- 5000 (gel filtration). This indicates that they are monomeric proteins sharing the same protein molecule. 3. AcPase III and IV differ essentially in thermostability and the activating effect of ConA binding. 4. AcPase III and IV are considerably activated with DTT but they differed markedly by the extent of this activation and the accompanying changes of their pH-activity curves. 5. It is suggested that the frog liver LMW AcPase represents a set of glycoforms whose different bioactivity is determined by the redox states of their essential cysteine residues.