Bacterial Acid Phosphatases Research Articles

Phosphorylation and dephosphorylation of polyhydroxy compounds by class A bacterial acid phosphatases.

Nonspecific acid phosphatases share a conserved active site with mammalian glucose-6-phosphatases (G6Pase). In this work we examined the kinetics of the phosphorylation of glucose and dephosphorylation of glucose-6-phosphate (G6P) catalysed by the acid phosphatases from Shigella flexneri (PhoN-Sf) and Salmonella enterica (PhoN-Se). PhoN-Sf is able to phosphorylate glucose regiospecifically to G6P, glucose-1-phosphate is not formed. The K(m) for glucose using pyrophosphate (PPi) as a phosphate donor is 5.3 mM at pH 6.0. This value is not significantly affected by pH in the pH region 4-6. The K(m) value for G6P by contrast is much lower (0.02 mM). Our experiments show these bacterial acid phosphatases form a good model for G6Pase. We also studied the phosphorylation of inosine to inosine monophosphate (IMP) using PPi as the phosphate donor. PhoN-Sf regiospecifically phosphorylates inosine to inosine-5'-monophosphate whereas PhoN-Se produces both 5'IMP and 3'IMP. The data show that during catalysis an activated phospho-enzyme intermediate is formed that is able to transfer its phosphate group to water, glucose or inosine. A general mechanism is presented of the phosphorylation and dephosphorylation reaction catalysed by the acid phosphatases. Considering the nature of the substrates that are phosphorylated it is likely that this class of enzyme is able to phosphorylate a wide range of hydroxy compounds.

Conserved proline residues near the N-terminus are important for enzymatic activity of class A bacterial acid phosphatases.

Conserved proline residues near the N-terminus are important for enzymatic activity of class A bacterial acid phosphatases.

Shigella apyrase – a novel variant of bacterial acid phosphatases?

A virulence-associated ATP diphosphohydrolase activity in the periplasm of Shigella, identified as apyrase, was found to be markedly similar to bacterial non-specific acid phosphatases in primary structure. When the Shigella apyrase sequence was threaded in to the recently published 3D structure of the highly similar (73%) Escherichia blattae acid phosphatase it was found to have a highly overlapping 3D structure. Our analysis, which included assays for phosphatase, haloperoxidase and catalase activities, led us to hypothesize that Shigella apyrase might belong to a new class of pyrophosphatase originating as one more variant in the family of bacterial non-specific acid phosphatases. It revealed interesting structure–function relationships and probable roles relevant to pathogenesis.

Haloperoxidase activity of bacterial acid phosphatases

Haloperoxidase activity of bacterial acid phosphatases

Legionella pneumophila major acid phosphatase and its role in intracellular infection.

Legionella pneumophila is an intracellular pathogen of protozoa and alveolar macrophages. This bacterium contains a gene (pilD) that is involved in both type IV pilus biogenesis and type II protein secretion. We previously demonstrated that the PilD prepilin peptidase is crucial for intracellular infection by L. pneumophila and that the secreted pilD-dependent proteins include a metalloprotease, an acid phosphatase, an esterase/lipase, a phospholipase A, and a p-nitrophenyl phosphorylcholine hydrolase. Since mutants lacking type IV pili, the protease, or the phosphorylcholine hydrolase are not defective for intracellular infection, we sought to determine the significance of the secreted acid phosphatase activity. Three mutants defective in acid phosphatase activity were isolated from a population of mini-Tn10-mutagenized L. pneumophila. Supernatants as well as cell lysates from these mutants contained minimal acid phosphatase activity while possessing normal levels of other pilD-dependent exoproteins. Genetic studies indicated that the gene affected by the transposon insertions encoded a novel bacterial histidine acid phosphatase, which we designated Map for major acid phosphatase. Subsequent inhibitor studies indicated that Map, like its eukaryotic homologs, is a tartrate-sensitive acid phosphatase. The map mutants grew within macrophage-like U937 cells and Hartmannella amoebae to the same degree as did wild-type legionellae, indicating that this acid phosphatase is not essential for L. pneumophila intracellular infection. However, in the course of characterizing our new mutants, we gained evidence for a second pilD-dependent acid phosphatase activity that, unlike Map, is tartrate resistant.

Secretion of an acid phosphatase (SapM) by Mycobacterium tuberculosis that is similar to eukaryotic acid phosphatases.

Mycobacterium tuberculosis secretes a large number of polypeptides with broad biological and immunological functions. We describe here the characterization of a 28-kDa acid phosphatase of M. tuberculosis (SapM) localized to the culture filtrate. The mature protein demonstrated biochemical characteristics similar to those of the bacterial nonspecific acid phosphatases. However, SapM yielded significant sequence homology to fungal acid phosphatases and not those of bacteria. Thus, SapM may represent a new class of bacterial nonspecific acid phosphatases.

Isolation, cloning, and expression of an acid phosphatase containing phosphotyrosyl phosphatase activity from Prevotella intermedia.

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.

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

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 [FILMAVT]-D-[ILFRMVY]-D-[GSNDE]-[TV]-[ILVAM]-[AT S VILMC]-X-¿YFWHKR)-X-¿YFWHNQ¿-X( 102,191)-¿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.

Bacterial acid phosphatase gene fusions useful as targets for cloning-dependent insertional inactivation.

The Morganella morganii phoC gene, encoding a class A acid phosphatase, was used to generate gene fusions with modified amino-terminal moieties of the Escherichia coli lacZ gene carrying a multiple-cloning site flanked by phage-specific promoters and recognition sites for universal sequencing primers. The corresponding hybrid proteins retained a PhoC-like enzymatic activity which is easily detectable by a plate histochemical assay, rendering similar gene fusions potentially useful as targets for cloning-dependent insertional inactivation. Cloning experiments performed in plasmids carrying similar lacZ-phoC fusions confirmed their usefulness as cloning vectors for direct screening of recombinants. As compared to conventional lacZ alpha-complementation-based vectors, which can only be used in E. coli hosts carrying specific lacZ mutations, the lacZ-phoC fusion-based vectors can be used in combination with any E. coli host and require a less expensive histochemical assay for screening of recombinants, while retaining all the advantageous features that made the former so popular as general purpose cloning vehicles.

An unexpected structural relationship between integral membrane phosphatases and soluble haloperoxidases.

The mechanism of a membrane-bound enzyme important in phospholipid signaling, type 2 phosphatidic acid phosphatase, is suggested by sequence motifs shared with a soluble vanadium-dependent chloroperoxidase of known structure. These regions are also conserved in other soluble globular and membrane-associated proteins, including bacterial acid phosphatases, mammalian glucose-6-phosphatases, and the Drosophila developmental protein Wunen. This implies that a similar arrangement of catalytic residues specifies the active site within both soluble and membrane spanning domains.

Immunological studies on the antitumor components of the basidiocarps ofAgrocybe cylindracea

The effects of cylindan, a polysaccharide isolated from the basidiocarps ofAgrocybe cylindracea, on murine sarcoma 180 tumor and murine immune cells were examined after intraperitoneal administration. Cylindan exhibited a marked life extension effect in mice against ascite forms of sarcoma 180 and Lewis lung carcinoma at a dose of 50 mg/kg/day, although it did not show any direct cytotoxicity against sarcoma 180, X5563, and MM46 murine tumor cells. Cylindan increased numbers of bone marrow stem cells as well as peritoneal exudate cells in flow cytometry using monoclonal antibodies. The tumor bearing mice group apparently showed the increase of macrophages and cytotoxic T lymphocytes in mouse spleen cells during the early stage of tumor growth. But during the later stage, the control group decreased immune cells and cylindan restored the decreased immune cells in the tumor bearing mice to the normal level. In non-specific immune response, cylindan stimulated the bacterial phagocytosis and acid phosphatase production in macrophages. It also activated components of the alternative complement pathway and natural killer activity against YAC-1 lymphoma. In humoral immunity, cylindan had a mitogenic effect against splenocytes and increased the number of plasma cells as token of stimulation of the differentiation of B lymphocytes. In cellular immunity, cylindan restored the depressed response of delayed type hypersensitivity in the tumor bearing mice to 60% of the normal level and increased the interleukin-2 (IL-2) responsiveness in the IL-2 dependent CTLL-2 cells. These results suggest that cylindan did not show direct cytotoxic effects on tumor cells but restored the decreased immune response of the tumor bearing mice.

Identification of a novel phosphatase sequence motif.

We have identified a novel, conserved phosphatase sequence motif, KXXXXXXRP-(X12-54)-PSGH-(X31-54)-SRXXXXX HXXXD, that is shared among several lipid phosphatases, the mammalian glucose-6-phosphatases, and a collection of bacterial nonspecific acid phosphatases. This sequence was also found in the vanadium-containing chloroperoxidase of Curvularia inaequalis. Several lines of evidence support this phosphatase motif identification. Crystal structure data on chloroperoxidase revealed that all three domains are in close proximity and several of the conserved residues are involved in the binding of the cofactor, vanadate, a compound structurally similar to phosphate. Structure-function analysis of the human glucose-6-phosphatase has shown that two of the conserved residues (the first domain arginine and the central domain histidine) are essential for enzyme activity. This conserved sequence motif was used to identify nine additional putative phosphatases from sequence databases, one of which has been determined to be a lipid phosphatase in yeast.

Cloning and characterization of the NapA acid phosphatase/phosphotransferase of Morganella morganii: identification of a new family of bacterial acid-phosphatase-encoding genes

The gene encoding a minor phosphate-irrepressible acid phosphatase (named NapA) of Morganella morganii was cloned and sequenced, and its product characterized. NapA is a secreted acid phosphatase composed of four 27 kDa polypeptide subunits. The enzyme is active on several organic phosphate monoesters but not on diesters, and is also endowed with transphosphorylating activity from organic phosphoric acid esters to nucleosides and other compounds with free hydroxyl groups. Its activity is inhibited by EDTA, inorganic phosphate, nucleosides and Ca2+, but not by fluoride or tartrate, and is enhanced by Mg2+, Co2+ and Zn2+. At the sequence level, the NapA enzyme did not show similarities to any other sequenced bacterial phosphatases. However, a search for homologous genes in sequence databases allowed identification of two open reading frames located within sequenced regions of the Escherichia coli and Proteus mirabilis genomes respectively, encoding proteins of unknown function which are highly homologous to the Morganella enzyme. Moreover, the properties of the NapA enzyme are very similar to those reported for the periplasmic nonspecific acid phosphatase II of Salmonella typhimurium (for which no sequence data are available). These data point to the existence of a new family of bacterial acid phosphatases, which we propose designating class B bacterial acid phosphatases.

Phosphatase production by a Citrobacter sp. growing in batch culture and use of batch cultures to investigate some limitations in the use of polyacrylamide gel-immobilized cells for product release

Phosphatase production by a Citrobacter sp. increased during exponential growth but was transiently repressed by glucose in medium containing glycerol 2-phosphate as the sole phosphorus source. In inorganic phosphate medium, phosphatase production was enhanced by carbon starvation, but was inhibited by an excess of glucose, or in glycerol-sufficient cultures upon entry into stationary phase. This was prevented by the provision of glycerol 2-phosphate as the phosphorus source. The data are consistent with known multiple regulatory patterns for other bacterial acid phosphatases and would account for interbatch variations in phosphatase titer. Such variation can be employed to prepare batches of cells of different phosphatase titer which can be used as tools to investigate the substrate diffusional limitations involved in the use of polyacrylamide gel-immobilized cells for phosphate release.

Circadian rhythm of acid phosphatase activity in rat liver microsomes and dependence of NADH 5 alpha-reductase on phosphatase activity.

The specific activity of acid phosphatase in male and female rats follows a circadian rhythm. Preincubation of liver microsomes with testosterone led to an increase of phosphatase activity and a loss of circadian rhythm. NADH 5 alpha-reductase was inactivated by several animal and bacterial acid and alkaline phosphatases while the acid phosphatase from potatoes was ineffective. The extent of inhibition depends on the course of circadian rhythm of NADH 5 alpha-reductase activity. Preincubation of microsomes in the presence of testosterone inhibited the NADH 5 alpha-reduction of testosterone. No such inhibition was observed after preincubation of microsomes with progesterone.

A new test to differentiate Serratia from Enterobacter.

This investigation has demonstrated a new indigogenic method for detecting production of bacterial acid phosphatase. The principle of the reaction is that bacterial acid phosphatase hydrolyzes an indolyl substrate, with the formation of a highly insoluble blue-green indigo which is deposited on the bacterial growth. The test differentiates pathogenic from nonpathogenic Staphylococci by demonstrating that pathogenic Staphylococci produce acid phosphatase. Differentiaton of Serratia from the other members in the Klebsiella-Enterobacter-Serratia group of organisms is difficult because of cultural similarities on blood and eosin methylene blue agar. Thus, a rapid test has been needed to differentiate Serratia from Enterobacter. Serratia produces the blue-green indigo color within 5 min., whereas Enterobacter produces the blue-green color in half an hour.

Multiple hydrolases in bean leaves (Phaseolus vulgaris L.) and the effect of the halo blight disease caused by Pseudomonas phaseolicola (Burkh.) Dowson.

Multiple forms of hydrolytic enzymes were demonstrated in extracts of healthy bean leaves (Phascolus vulgaris L.) and bean leaves infected with the halo blight organism [Pseudomonas phaseolicola (Burkh.) Dowson] by polyacrylamide disc electrophoresis. Bean leaves contained up to 4 acid phosphatase bands, 9 esterase bands active towards alpha-naphthyl acetate, and 7 esterase bands towards alpha-naphthyl butyrate. Only low or no activity was found for alkaline phosphatase, lipase, and aminopeptidase. Two artifacts are described which were observed with the lead phosphate method for acid phosphatase and the Tween method for demonstration of lipase. After infection with the halo blight organism the major acid phosphatase of the host increased during early and decreased at later infection stages. An acid phosphatase of bacterial origin with a more neutral pH optimum could be demonstrated in infected leaves. It is suggested that the bacterial acid phosphatase plays a role in uptake of metabolites by the pathogen. Several esterase bands decreased after infection. One host band with activity towards alpha-naphthyl butyrate increased. Also the pathogen showed an esterase band with high activity towards alpha-naphthyl butyrate.

Influence of inorganic phosphate in the formation of phosphatases by Escherichia coli

Both acid and alkaline phosphatase have been demonstrated in strains of E. coli. Bacterial acid phosphatase (pH optimum 4–5) displays a fairly high specificity for the hexosephosphates, while the alkaline enzyme (pH optimum 8.5–9.5) hydrolyzes all the phosphomonoesters tested. The kinetics of formation of both enzymes have been studied and it has been shown that alkaline phosphatase in measurable amount is only formed when Pi becomes limiting in the medium, at which point the enzyme is formed in substantial amount at a maximum rate. The implications of the findings with respect to mechanisms of controlling enzyme formation are discussed.