Expression, purification, crystallization and preliminary X-ray diffraction studies of recombinant class B non-specific acid phosphatase ofSalmonella typhimurium

Abstract

The aphA gene of Salmonella enterica sv. Typhimurium strain MD6001 was cloned in the multicopy plasmid pBluescript SK(-). The recombinant AphA protein was purified to homogeneity. The protein crystallized in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 112.4, b = 130.2, c = 139.6 A. Consistent with the self-rotation function, there are two tetramers in the asymmetric unit, indicating a solvent content of approximately 54%. The crystals are composed of biologically active AphA molecules.

Exploiting Acid Phosphatases in the Synthesis of Phosphorylated Monoalcohols and Diols

“…Acid phosphatase phytase catalyzes the hydrolysis of phytate, a phosphorylated natural cyclitol by liberating inorganic phosphate and is therefore commonly used as animal feedstock additive 20. Acid phosphatase AphA‐St from Salmonella typhimurium LT221 was selected due to its phosphotransferase activity on alcohols using p ‐NPP as donor 22. PiACP from Prevotella intermedia 23 was selected based on high sequence similarity with robust PhoC‐Mm developed for nucleoside phosphorylation 24.…”

“…Strong hydrolytic activity on inorganic phosphate donors was still observed, confirming active protein (data not shown). AphA‐St also contains Mg 2+ (in addition to the catalytically active nucleophile aspartate21), whose activity is affected by inorganic polyphosphates by metal chelation. Although the addition of 10 m M MgCl 2 could partly restore the hydrolytic activity on p ‐NPP in the presence of 250 m M PP i (Figure S2), only traces of product and marginal hydrolytic activity were obtained in the transphosphorylation of 1,4‐butanediol (500 m M ) by using 100 m M PP i as donor at pH = 4.2 and 5.5, independent of the MgCl 2 concentration (10, 50, 100 m M ) (data not shown).…”

Assimilation of Nicotinamide Mononucleotide Requires Periplasmic AphA Phosphatase inSalmonella enterica

“…The affected sequence was perfectly conserved in Escherichia coli and S. enterica and shares features with known DeoR repressor binding sites in the E. coli genome. Because the AphA enzyme removes phosphate from nucleotides and is known to be important for nucleotide assimilation (24,45), it seemed reasonable that aphA might be repressed by DeoR, a regulator of genes involved in use of deoxynucleotides as carbon sources. However, a constructed deoR deletion mutation caused no discernible improvement in the ability to grow on NMN, suggesting that the AphA* mutations relieve aphA repression by some other regulatory protein.…”

“…The AphA protein used by Salmonella is a nonspecific periplasmic acid phosphatase (40) that contributes to assimilation of various purine and pyrimidine nucleotides by removing phosphate (24,45) and thus does not appear to be dedicated to uptake of pyridines. Wild-type S. enterica appears to obtain pyridines by uptake of either free nicotinic acid, Nm, or (via PnuC) NmR (all of which are used at external concentrations below 1 M).…”