ABSTRACT
MDP-1 is a eukaryotic magnesium-dependent acid phosphatase with little sequence homology to previously characterized phosphatases. The presence of a conserved motif (Asp-X-Asp-X-Thr) in the N terminus of MDP-1 suggested a relationship to the haloacid dehalogenase (HAD) superfamily, which contains a number of magnesium-dependent acid phosphatases. These phosphatases utilize an aspartate nucleophile and contain a number of conserved active-site residues and hydrophobic patches, which can be plausibly aligned with conserved residues in MDP-1. Seven site-specific point mutants of MDP-1 were produced by modifying the catalytic aspartate, serine, and lysine residues to asparagine or glutamate, alanine, and arginine, respectively. The activity of these mutants confirms the assignment of MDP-1 as a member of the HAD superfamily. Detailed comparison of the sequence of the 15 MDP-1 sequences from various organisms with other HAD superfamily sequences suggests that MDP-1 is not closely related to any particular member of the superfamily. The crystal structures of several HAD family enzymes identify a domain proximal to the active site responsible for important interactions with low molecular weight substrates. The absence of this domain or any other that might perform the same function in MDP-1 suggests an "open" active site capable of interactions with large substrates such as proteins. This suggestion was experimentally confirmed by demonstration that MDP-1 is competent to catalyze the dephosphorylation of tyrosine-phosphorylated proteins.
Subject(s)
Aspartic Acid/chemistry , Phosphoprotein Phosphatases/chemistry , Sequence Homology, Amino Acid , Amino Acid Motifs/genetics , Amino Acid Sequence , Animals , Catalytic Domain/genetics , Humans , Hydrolases/chemistry , Hydrolases/genetics , Mice , Molecular Sequence Data , Multigene Family , Mutagenesis, Site-Directed , Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/metabolism , Protein Phosphatase 2C , Protein Structure, Tertiary/genetics , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/metabolism , Rats , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Sequence AlignmentABSTRACT
We report here the purification, cloning, expression, and characterization of a novel phosphatase, MDP-1. In the course of investigating the reported acid phosphatase activity of carbonic anhydrase III preparations, several discrete phosphatases were discerned. One of these, a magnesium-dependent species of 18.6 kDa, was purified to homogeneity and yielded several peptide sequences from which the parent gene was identified by database searching. Although orthologous genes were identified in fungi and plants as well as mammalian species, there was no apparent homology to any known family of phosphatases. The enzyme was expressed in Escherichia coli with a fusion tag and purified by affinity methods. The recombinant enzyme showed magnesium-dependent acid phosphatase activity comparable to the originally isolated rabbit protein. The enzyme catalyzes the rapid hydrolysis of p-nitrophenyl phosphate, ribose-5-phosphate, and phosphotyrosine. The selectivity for phosphotyrosine over phosphoserine or phosphothreonine is considerable, but the enzyme did not show activity toward five phosphotyrosine-containing peptides. None of the various substrates assayed (including various nucleotide, sugar, amino acid and peptide phosphates, phosphoinositides, and phosphodiesters) exhibited K(M) values lower than 1 mM, and many showed negligible rates of hydrolysis. The enzyme is inhibited by vanadate and fluoride but not by azide, cyanide, calcium, lithium, or tartaric acid. Chemical labeling, refolding, dialysis, and mutagenesis experiments suggest that the enzymatic mechanism is not dependent on cysteine, histidine, or nonmagnesium metal ions. In recognition of these observations, the enzyme has been given the name magnesium-dependent phosphatase-1 (MDP-1).
Subject(s)
Phosphoprotein Phosphatases/genetics , Amino Acid Sequence , Animals , Catalysis , Cations , Chromatography, Affinity , Cloning, Molecular , Cysteine/metabolism , Enzyme Inhibitors/pharmacology , Histidine/metabolism , Hydrogen-Ion Concentration , Magnesium/metabolism , Mice , Molecular Sequence Data , Phosphoprotein Phosphatases/chemistry , Phosphoprotein Phosphatases/metabolism , Protein Phosphatase 1 , Protein Phosphatase 2C , Rabbits , Sequence Analysis, Protein , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
PURPOSE: To use light microscopy to observe the urease-induced growth of struvite crystals in real-time, and to compare the effects of various proteins on that growth. MATERIALS AND METHODS: Artificial urine, with and without citrate, and a minimal urine solution containing only urea and the components of struvite and apatite were incubated with urease and test proteins in the depressions of culture slides. The number and size of rectangular and X-shaped struvite crystals were recorded using a low-power phase contrast microscope. RESULTS: The formation of crystalline struvite appears to occur after the formation of an amorphous calcium- and magnesium-containing phase. The extent of this amorphous phase is dependent on the presence of calcium and citrate, both of which strongly promote its formation over the crystalline phase. alpha-globulin, gamma-globulin and chymotrypsin inhibitor all result in the same amount of crystalline struvite as bovine serum albumin which is used as a control. Calprotectin, on the other hand, causes consistent and significant reductions in the number and size of struvite crystals under a wide range of conditions. No changes in the morphology of the struvite crystals were observed. CONCLUSIONS: Calprotectin, the dominant protein of infection stone matrix, has distinctive properties which affect the formation and growth of struvite crystals. The presence of citrate in synthetic urine dramatically reduces the number of struvite crystals observed. The present method for observing the effects of putative infection stone inhibitors appears to have merit.
