Heterologous expression and characterization of recombinant purple acid phosphatase from red kidney bean

Purple acid phosphatases (PAPs) are dinuclear metallohydrolases of widespread occurrence. In a first step to understand structure-function relationship of PAP from red kidney bean (kbPAP), we cloned its cDNA and functionally expressed the enzyme in insect cells. kbPAP cDNA encodes a protein of 459 amino acids with 99% identity to the published primary structure (T. Klabunde et al., Eur. J. Biochem. 226 (1994) 369-375). N-terminally the cDNA encodes 27 amino acids with characteristics for a signal directing the nascent protein to the endoplasmic reticulum. A baculovirus vector was constructed containing cDNAs of kbPAP and green fluorescent protein, the latter to serve as transfection and infection marker. Heterologous expression in High Five insect cells afforded a dimeric, disulfide-linked phosphatase of 110 kDa, identical to the mass of native kbPAP. Purification in three steps yielded 1.5 mg recombinant protein per liter of culture medium with a specific activity of 266 units/mg, slightly exceeding that of native kbPAP. The recombinant protein was functionally indistinguishable from native kbPAP, despite differences in glycosylation and sensitivity to redox reagents.     

Crystallization and preliminary crystallographic data of purple acid phosphatase from red kidney bean.

Purple acid phosphatase from red kidney bean has been crystallized from ammonium sulfate solutions in the pH range from 3.5 to 5.5. The crystal form is tetragonal bipyramidal and the largest crystals grew up to 2.0 mm long. Systematic absences indicate one of the enantiomorphic space groups P4(1)2(1)2 (92) or P4(3)2(1)2 (96) with cell dimensions a = b = 104.1(1) A and c = 308.7(2) A. The asymmetric unit contains one dimer with Mr of 110,700, determined by ultraviolet-laser desorption mass spectrometry. The crystals, with a salt-free density of 1.12 g/cm3 and a water content of 67%, diffract to 3.5 A.     

Derivatives of the purple phosphatase from red kidney bean: Replacement of zinc with other divalent metal ions

Apoenzyme, containing ⩽0.1 zinc atoms and ⩽0.2 Fe atoms per subunit and with ⩽3% of the phosphatase activity, has been prepared from native red kidney bean purple phosphatase. Treatment of this apoenzyme with Fe³⁺ or Zn²⁺ separately gave very little recovery of activity, whereas treatment with both Fe³⁺ and Zn²⁺ resulted in complete restoration of activity, indicating that both metal ions are essential. ZnFe enzyme with close to one iron and one zinc atom per subunit has been reconstituted by this procedure. Essentially full reactivation was also achieved by addition of Fe³⁺ together with Fe²⁺ or Co²⁺ to the apoenzyme; Fe³⁺ and Cd²⁺ gave 27% restoration of activity, whereas Fe³⁺ with Mn²⁺, Cu²⁺, Ni²⁺ or Hg²⁺ gave little or no increase in activity. Kinetic parameters for the hydrolysis of p-nitrophenyl phosphate and ATP by the FeFe derivative are reported.     

Structure-function relationships of purple acid phosphatase from red kidney beans based on heterologously expressed mutants

Purple acid phosphatases are binuclear metalloenzymes, which catalyze the conversion of orthophosphoric monoesters to alcohol and orthophosphate. The enzyme from red kidney beans is characterized with a Fe(III)-Zn(II) active center. So far, the reaction mechanisms postulated for PAPs assume the essentiality of two amino acids, residing near the bimetallic active site. Based on the amino acid sequence of kidney bean PAP (kbPAP), residues H296 and H202 are believed to be essential for catalytic function of the enzyme. In the present study, the role of residue H202 has been elucidated. Mutants H202A and H202R were prepared by site-directed mutagenesis and expressed in baculovirus-infected insect cells. Based on kinetic studies, residue H202 is assumed to play a role in stabilizing the transition state, particularly in charge compensation, steric positioning of the substrate, and facilitating the release of the product by protonating the substrate leaving groups. The study confirmed the essentiality and elucidates the functional role of H202 in the catalytic mechanism of kbPAP.     

Characterization of a novel acid phosphatase from embryonic axes of kidney bean exhibiting vanadate-dependent chloroperoxidase activity

A novel colorless acid phosphatase (KeACP), which was distinct from the kidney bean purple acid phosphatase, was purified to apparent homogeneity and cloned from embryonic axes of kidney bean (Phaseolus vulgaris L. Ohfuku) during germination. When orthovanadate (VO(4)(-3)) is added to the apo form of the enzyme, KeACP uniquely exhibits the chloroperoxidase activity with loss of phosphatase activity. This is the first demonstration that KeACP is a vanadate-dependent chloroperoxidase in plants to be characterized and suggests that KeACP may play a role in modifying a wide variety of chlorinated compounds that are present in higher plants. The enzyme is a dimer that presents three forms made up of the combination of the dominant 56-kDa and the minor 45-kDa subunits, and both subunits contain carbohydrate. The full-length cDNA of the KeACP gene is 1641 nucleotides, and this sequence is predicted to encode a protein having 457 amino acid residues (52,865 Da), including a signal peptide. The complete nucleotide sequence of the genomic DNA (3228 bp) of KeACP consists of seven exons and six introns. Northern blot analysis demonstrated that the KeACP gene was expressed specifically in embryonic axes of the kidney bean, and its expression coincided with elongation of the embryonic axis during germination.     

Bovine kidney alkaline phosphatase. Purification, subunit structure, and metalloenzyme properties.

Kidney alkaline phosphatase was purified to homogeneity. It is a glycoprotein of about 172,000 molecular weight. Analyses of the subunit structure by sedimentation equilibrium in 6 M guanidine hydrochloride and by gel electrophoresis in sodium dodecyl sulfate indicate that the alkaline phosphatase is a dimer comprising two very similar or identical subunits of about 87,000 molecular weight. The native enzyme contains 4.5 +/- 0.2 g atoms of zinc per mol of protein. Reconstitution experiments from the apophosphatase show that binding of 4 Zn2+ per mol of dimer is essential for full activity. The kinetic data of Zn2+ binding to the apoprotein require at least a two-step mechanism, in which one of the steps corresponds to a conformational change within the enzyme. This paper also presents data concerning amino acid composition, sugar content, enzyme stability, absorbance index, and sedimentation velocity.     

An EXAFS study of jack bean urease,a nickel metalloenzyme

EXAFS and XANES spectra have been recorded above the nickel K edge of urease and three model compounds. Preliminary results indicate that the local environment of the nickel ions in urease resemble most closely that of the nickel ions in the model compound [Ni(L)₂(L)₁] (ClO₄)₁, where L is 1-n-propyl-2-α-hydroxybenzyl benzimidazole and L is the deprotonated form.     

Purification and characterization of a purple acid phosphatase from rat spleen

An acid phosphatase species which is activated by Fe2+ was purified 3,700-fold from rat spleen by chromatography on columns containing Blue-Sepharose, concanavalin A-Sepharose, Sephadex G-100, and CM-Sephadex. The enzyme hydrolyzed aryl phosphates, nucleoside di- and triphosphates, phosphoproteins, and thiamine pyrophosphate with Km values of 10(-4) to 10(-3) M at an optimal pH of 5.0-5.8. Co-purification of the acid phosphatase and acid phosphoprotein phosphatase indicated that they were identical. The purified enzyme was glycoprotein in nature, showing four heterogeneous forms on acid polyacrylamide gel electrophoresis (pI values, 7.8, 8.0, 8.3, and 8.5), but it gave a molecular weight of 33,000 on sodium dodecyl sulfate-gel electrophoresis and gel permeation chromatography. The enzyme had a purple color (lambda max 545 nm) and contained 2 iron atoms per enzyme molecule. Among reductants, ascorbic acid and Fe2+ were the best activators, although their combined effect was not additive. Fe2+ and ascorbic acid both changed the purple enzyme into the same active form (lambda max 515 nm), giving almost the same kinetic constants for substrates and for inhibitors such as molybdate, phosphate and fluoride. However, low concentrations of Fe2+, from 0.01 mM to 1.0 mM, immediately and reversibly activated the enzyme, whereas high concentrations of ascorbic acid over 1 mM were required for maximal activation, which was slow and irreversible.     

Purification of a cellulase from kidney bean abscission zones

The purification of a cellulase isoenzyme with a pI of 9.5 from kidney bean abscission zones is described. An important step in the purification involved the adsorption of the cellulase isoenzyme onto an affinity column of CF-11 cellulose and the subsequent elution with cellobiose. Native and SDS polyacrylamide gel electrophoresis established that there was only one component in the purified cellulase samples. Antibodies raised against the purified pI 9.5 cellulase precipitated this isoenzyme from crude or purified solutions but did not cross react with pI 4.5 cellulase from 2,4-D-treated abscission zones. The antibody was shown to be monospecific by immunoelectrophoresis and by the fact that it precipitated only a single ¹⁴C-labeled protein from an abscission zone extract heavily labeled with ¹⁴C amino acids.     

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.     

Purification and characterization of a secreted purple phosphatase from soybean suspension cultures

We purified and partially sequenced a purple (λ_max = 556 nanometers) acid phosphatase (APase; EC 3.1.3.2) secreted by soybean (Glycine max) suspension-culture cells. The enzyme is a metalloprotein with a Mn²⁺ cofactor. This APase appears to be a glycoprotein with a monomer subunit molecular weight of 58,000 and an active dimer molecular weight of approximately 130,000. The protein has an isoelectric point of about 5.0 and a broad pH optimum centered near 5.5. The purified enzyme, assayed with p-nitrophenyl phosphate as the substrate, has a specific activity of 512 units per milligram protein and a K_m of approximately 0.3 millimolar; phosphate is a competitive inhibitor with a K_i of 0.7 millimolar. This APase is similar to one found in soybean seed meal but dissimilar to that found in soybean seedlings.     

Formaldehyde dehydrogenase from Pseudomonas putida: a zinc metalloenzyme

The NAD+-dependent formaldehyde dehydrogenase from Pseudomonas putida C-83 was found to contain 4 gram atoms of zinc per mol, corresponding to 2 gram atoms of zinc per subunit monomer. Treatment of the enzyme with o-phenanthroline resulted in removal of 1 gram atom of zinc per subunit and caused a complete inactivation of the enzyme. The activity lost was restored by the addition of zinc ions, by which the zinc content was also reversed to almost the same level as that of the native enzyme. Another zinc atom that was resistant to metal chelator-treatment was liberated from the enzyme only after the irreversible denaturation of the enzyme. These results indicate that the formaldehyde dehydrogenase of P. putida is a zinc metalloenzyme and one of two zinc atoms per subunit participates in the catalytic activity of the enzyme, another zinc being presumably involved in maintaining the native conformation of the enzyme. Treatment of the enzyme with bipyridine also caused a reversible inactivation of the enzyme, but the zinc content remained unchanged. The spectrophotometric analysis indicated that the formation of a enzyme-Zn-bipyridine complex took place. Incubation of the enzyme with p-chloromercuribenzoate also resulted in a complete loss of the activity. These results suggest that an intrinsic zinc and sulfhydryl group together with NAD+ participate in the dehydrogenation reaction of substrate by the enzyme.     

Purification and characterization of a protein phosphatase from winged bean.

A protein phosphatase (WbPP) has been purified from the soluble fraction of the winged bean (Psophocarpus tetragonolobus) shoot extract. The preparation is essentially homogenous as shown by the constant specific activity of the enzyme across the peak fractions, eluted from the thiophosphorylated histone-Sepharose affinity column, the last step of purification and by single protein bands on polyacrylamide gel electrophoresis (PAGE) in the presence as well as absence of denaturating agents. The monomeric nature of WbPP is revealed by an M(r) of 92,000 and 85,000, respectively, as estimated by SDS-PAGE and gel permeation chromatography under non-denaturating conditions. Autophosphorylated calmodulin-like domain protein kinase (P-WbCDPKI) [Saha, P., & Singh, M. (1995). Biochem. J., 305, 205] and phosphohistone H1 (P-hisH1), prepared by using the other homologous CDPK, i.e. WbCDPKII [Ganguly, S., & Singh, M. (1998). Phytochemistry, 48(1), 61], are good substrates of the purified enzyme, while P-hisH1 and phosphocasein prepared by using heterologous cAMP-dependent protein kinase, are respectively very poor and totally inactive as substrate. WbPP is adjudged to be a protein phosphoserine phosphatase since phosphoserine is the only phosphorylated amino acid residue detected in our earlier analysis of P-WbCDPKI and P-hisH1. The enzyme is strongly stimulated by a combination of Mg2+ and Ca2+, without being dependent on either of them and is also unaffected by calmodulin and fluphenazine. Orthovanadate strongly inhibits the enzyme while okadaic acid is a poor inhibitor.     

Superoxide dismutases from kidney bean leaves

Three isozymes of superoxide dismutase were found in the solubleextract of kidney bean leaves. Two of them, purified to nearhomogeneous states, were inhibited by cyanide and by the antibodyto spinach Cu, Zn-superoxide dismutase. Thus, both isozymeswere the Cu and Zn containing enzyme that has a molecular weightof 32,000 and is composed of two subunits of equal size. Anotherisozyme was insensitive to cyanide and to the antibody, butwas inactivated by acetone or heating. The cyanideinsensitiveisozyme was not inactivated by H₂O₂ showing that this isozymeis Mn-superoxide dismutase. (Received June 13, 1979; )     

Specific interaction of human Tamm-Horsfall gylcoprotein with leucoagglutinin, a lectin from Phaseolus vulgaris (red kidney bean).

Human Tamm-Horsfall glycoprotein inhibits lymphocyte transformation induced by leucoagglutinin and haemagglutinin from Phaseolus vulgaris (red kidney bean). The glycoprotein interacts with the two lectins, giving insoluble precipitates. The interaction with leucoagglutinin is highly specific, and the shape of the precipitin curve is that of an antigen-antibody reaction; precipitation is specifically inhibited by N-acetyl-D-galactosamine. Results are discussed, and it is suggested that inhibition of lymphocyte transformation is due to competition between human Tamm-Horsfall glycoprotein and carbohydrate receptors on lymphocytes for the two lectins. The interaction between human Tamm-Horsfall glycoprotein and Phaseolus vulgaris lectins has been used to develop a one-step procedure for the separation of the two lectins by affinity chromatography on (human Tamm-Horsfall-glycoprotein)-Sepharose.     

Binding of isolectins from red kidney bean (Phaseolus vulgaris) to purified rat brush-border membranes

Ingestion of red kidney bean phytohemagglutinin causes impaired growth and intestinal malabsorption, and facilitates bacterial colonization in the small intestine of weanling rats. We have studied interactions of the highly purified phytohemagglutinin erythroagglutinating (E4) and mitogenic (L4) isolectins with microvillous membrane vesicles prepared from rat small intestines. E4 and L4 were radioiodinated with 125I by the chloramine-T technique. E4 and L4 isolectins both bound to microvillous membrane vesicles. Binding was saturable and reversible. Each mg of membrane protein bound 744 +/- 86 micrograms E4 and 213 +/- 21 micrograms L4. The apparent Ka for E4 and L4 binding was 2.5 x 10(-6) and 13.0 x 10(-6) M-1, respectively. Binding of each 125I-labelled isolectin was abolished by 100-fold excess of unlabelled isolectin. In each case binding also was inhibited by appropriate oligosaccharide inhibitors, indicating that isolectin-microvillous membrane interactions were mediated by carbohydrate recognition. Patterns of saccharide inhibition of isolectin binding were different for E4 and L4. Competitive binding experiments demonstrated mutual noncompetitive inhibition of E4 and L4 binding consistent with steric hindrance. Therefore, E4 and L4 each bound to its own set of receptors. Based on the known saccharide specificities of E4 and L4, these data indicate that there are differences in expression of complex asparagine-linked biantennary and tri- or tetraantennary oligosaccharides at the microvillous surface. The data also provide the possibility that direct interactions of one or more phytohemagglutinin isolectins with intestinal mucosa in vivo may contribute to the antinutritional effects associated with ingestion of crude red kidney beans.     

Crystal structure of a mammalian purple acid phosphatase.

Tartrate-resistant acid phosphatase (TRAP) is a mammalian di-iron- containing enzyme that belongs to the family of purple acid phosphatases (PAP). It is highly expressed in a limited number of tissues, predominantly in bone-resorbing osteoclasts and in macrophages of spleen. We have determined the crystal structure of rat TRAP in complex with a phosphate ion to 2.7 A resolution. The fold resembles that of the catalytic domain of kidney bean purple acid phosphatase (KBPAP), although the sequence similarity is limited to the active site residues. A surface loop near the active site is absent due to proteolysis, leaving the active-site easily accessible from the surrounding solvent. This, we believe, gives a structural explanation for the observed proteolytic activation of TRAP. The current structure was determined at a relatively high pH and without any external reducing agents. It is likely that it represents an oxidized and therefore catalytically inactive form of the enzyme.