Expression, purification and preliminary X-ray characterization of histidinol phosphate phosphatase.

Histidinol phosphate phosphatase (HisPPase) catalyzes the eighth step of histidine biosynthesis, in which L-histidinol phosphate undergoes dephosphorylation to give histidinol. A recombinant form of the histidinol phosphate phosphatase from Thermus thermophilus HB8 has been expressed in Escherichia coli, purified and crystallized in two crystal forms by the hanging-drop vapour-diffusion technique. Crystal form I belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 84.8, b = 97.2, c = 74.9 A, and crystal form II belongs to the orthorhombic space group C222(1), with unit-cell parameters a = 76.9, b = 157.6, c = 116.7 A. The crystals probably contain two monomers in the asymmetric unit, with V(M) values of 2.57 A(3) Da(-1) for form I and 2.96 A(3) Da(-1) for form II. X-ray data have been collected to 1.70 and 1.75 A resolution for crystal forms I and II, respectively.

Properties of a constitutive alkaline phosphatase from strain 74A of the mold Neurospora crassa.

A constitutive alkaline phosphatase was purified to apparent homogeneity as determined by polyacrylamide gel electrophoresis from mycelia of the wild strain 74A of the mold Neurospora crassa, after growth on acetate and in the presence of saturating amounts of inorganic phosphate (Pi) for 72 h at 30 degrees C. The molecular mass was 58 kDa and 56 kDa as determined by exclusion chromatography and SDS-PAGE, respectively. This monomeric enzyme shows an apparent optimum pH ranging from 9.5 to 10.5 and Michaelis kinetics for the hydrolysis of p-nitrophenyl phosphate (the K(m) and Hill coefficient values were 0.35 mM and 1.01, respectively), alpha-naphthyl phosphate (the K(m) and Hill coefficient values were 0.44 mM and 0.97, respectively), beta-glycerol phosphate (the K(m) and Hill coefficient values were 2.46 mM and 1.01, respectively) and L-histidinol phosphate (the K(m) and Hill coefficient values were 0.47 mM and 0.94, respectively) at pH 8.9. The purified enzyme is activated by Mg(2+), Zn(2+) and Tris-HCl buffer, and is inhibited by Be(2+), histidine and EDTA. Also, 0.3 M Tris-HCl buffer protected the purified enzyme against heat inactivation at 70 degrees C (half-life of 19.0 min, k = 0.036 min(-1)) as compared to 0.3 M CHES (half-life of 2.3 min, k = 0.392 min(-1)) in the same experiment.

Properties of a constitutive alkaline phosphatase from strain 74A of the mold Neurospora crassa

A constitutive alkaline phosphatase was purified to apparent homogeneity as determined by polyacrylamide gel electrophoresis from mycelia of the wild strain 74A of the mold Neurospora crassa, after growth on acetate and in the presence of saturating amounts of inorganic phosphate (Pi) for 72 h at 30ºC. The molecular mass was 58 kDa and 56 kDa as determined by exclusion chromatography and SDS-PAGE, respectively. This monomeric enzyme shows an apparent optimum pH ranging from 9.5 to 10.5 and Michaelis kinetics for the hydrolysis of p-nitrophenyl phosphate (the Km and Hill coefficient values were 0.35 mM and 1.01, respectively), alpha-naphthyl phosphate (the Km and Hill coefficient values were 0.44 mM and 0.97, respectively), ß-glycerol phosphate (the Km and Hill coefficient values were 2.46 mM and 1.01, respectively) and L-histidinol phosphate (the Km and Hill coefficient values were 0.47 mM and 0.94, respectively) at pH 8.9. The purified enzyme is activated by Mg2+, Zn2+ and Tris-HCl buffer, and is inhibited by Be2+, histidine and EDTA. Also, 0.3 M Tris-HCl buffer protected the purified enzyme against heat inactivation at 70ºC(half-life of 19.0 min, k = 0.036 min-1) as compared to 0.3 M CHES (half-life of 2.3 min, k = 0.392 min-1) in the same experiment

Histidinol phosphate phosphatase, catalyzing the penultimate step of the histidine biosynthesis pathway, is encoded by ytvP (hisJ) in Bacillus subtilis.

The deduced product of the Bacillus subtilis ytvP gene is similar to that of ORF13, a gene of unknown function in the Lactococcus lactis histidine biosynthesis operon. A B. subtilis ytvP mutant was auxotrophic for histidine. The only enzyme of the histidine biosynthesis pathway that remained uncharacterized in B. subtilis was histidinol phosphate phosphatase (HolPase), catalyzing the penultimate step of this pathway. HolPase activity could not be detected in crude extracts of the ytvP mutant, while purified glutathione S-transferase-YtvP fusion protein exhibited strong HolPase activity. These observations demonstrated that HolPase is encoded by ytvP in B. subtilis and led us to rename this gene hisJ. Together with the HolPase of Saccharomyces cerevisiae and the presumed HolPases of L. lactis and Schizosaccharomyces pombe, HisJ constitutes a family of related enzymes that are not homologous to the HolPases of Escherichia coli, Salmonella typhimurium, and Haemophilus influenzae.

Purification of the bifunctional enzyme, imidazoleglycerolphosphate dehydratase-histidinol phosphatase, of Salmonella typhimurium.

Mn2+ precipitation, antibody affinity chromatography, and selective proteolysis have been used to purify a bifunctional core of the hisB enzyme from Salmonella typhimurium. The core is homogeneous in subunit molecular weight; however, it is heterogeneous in its charge properties, probably as a result of multiple cleavage points produced by limited proteolytic digestion. The resistance of the enzyme to irreversible denaturation by urea allowed the use of urea to elute the hisB enzyme from a column of anti-hisB IgG immobilized on Sepharose. Heterogeneous oligomeric forms of the enzyme were demonstrated by electrophoretic analysis and exist as multiples of the 46,000 molecular weight monomer.