Characterization of heterodimeric alkaline phosphatases from Escherichia coli: an investigation of intragenic complementation.

Escherichia coli alkaline phosphatase (EC 3.1.3.1) belongs to a rare group of enzymes that exhibit intragenic complementation. When certain mutant versions of alkaline phosphatase are combined, the resulting heterodimeric enzymes exhibit a higher level of activity than would be expected based upon the relative activities of the parental enzymes. Nine previously identified alkaline phosphatase complementation mutants were re-examined in this work in order to determine a molecular explanation of intragenic complementation in this experimental system. The locations of these mutations were determined by DNA sequence analysis after PCR amplification of the phosphatase-negative phoA gene. Most of the mutations involved ligands to metal-binding sites. Each of the mutant enzymes was re-created by site-specific mutagenesis, expressed, purified, and kinetically characterized. To investigate cooperativity between the two subunits, we analyzed heterodimeric forms of some of the site-specific mutant enzymes. To enable the isolation of the heterodimeric alkaline phosphatase in pure form, the overall charge of one subunit was altered by replacing the C-terminal Lys residue with three Asp residues. This modification had no effect on the kinetic properties of the enzyme. Heterodimeric alkaline phosphatases were created using two methods: (1) in vitro formation by dissociation at acid pH followed by reassociation at slightly alkaline pH conditions in the presence of zinc and magnesium ions; and (2) in vivo expression from a plasmid carrying two different phoA genes. Increases in k(cat), as well as a large reduction in the p-nitrophenyl phosphate K(m) were observed for certain combinations of mutant enzymes. These results suggest that the structural assembly of E. coli alkaline phosphatase into the dimer induces cooperative interactions between the monomers necessary for the formation of the functional form of the holoenzyme.

Kinetic and X-ray structural studies of three mutant E. coli alkaline phosphatases: insights into the catalytic mechanism without the nucleophile Ser102.

Escherichia coli alkaline phosphatase (EC 3.1.3.1) is a non-specific phosphomonoesterase that catalyzes the hydrolysis reaction via a phosphoseryl intermediate to produce inorganic phosphate and the corresponding alcohol. We investigated the nature of the primary nucleophile, fulfilled by the deprotonated Ser102, in the catalytic mechanism by mutating this residue to glycine, alanine and cysteine. The efficiencies of the S102G, S102A and S102C enzymes were 6 x 10(5)-fold, 10(5)-fold and 10(4)-fold lower than the wild-type enzyme, respectively, as measured by the kcat/Km ratio, still substantially higher than the non-catalyzed reaction. In order to investigate the structural details of the altered active site, the enzymes were crystallized and their structures determined. The enzymes crystallized in a new crystal form corresponding to the space group P6322. Each structure has phosphate at each active site and shows little departure from the wild-type model. For the S102G and S102A enzymes, the phosphate occupies the same position as in the wild-type enzyme, while in the S102C enzyme it is displaced by 2.5 A. This kinetic and structural study suggests an explanation for differences in catalytic efficiency of the mutant enzymes and provides a means to study the nature and strength of different nucleophiles in the same environment. The analysis of these results provides insight into the mechanisms of other classes of phosphatases that do not utilize a serine nucleophile.

Cloning, overexpression, refolding, and purification of the nonspecific phospholipase C from Bacillus cereus.

Bacillus cereus secretes a nonspecific phospholipase C (PLC) that catalyzes the hydrolysis of phospholipids to yield diacylglycerol and a phosphate monoester. B. cereus PLC has been overexpressed with its signal sequence in Escherichia coli using a T7 expression system. The expressed enzyme formed intracellular inclusion bodies which were solubilized in the presence of 8 M urea. Renaturation was initiated by gradual removal of urea and addition of zinc ions. The signal peptide was specifically cleaved by a protease, clostripain, added when the urea concentration was 1.5 M. Factors that led to protein reaggregation included rapid removal of urea, use of Tris instead of barbital buffer, and presence of the signal peptide when the urea concentration was below 1.5 M. The folded protein was purified by Q-Sepharose Fast flow chromatography to yield a preparation > 99% pure. The final yield of active enzyme was 30-40 mg per liter of culture. The recombinant PLC exhibited biochemical and kinetic properties identical to those of extracellularly produced PLC from B. cereus. Site-specific mutagenesis of Asn-134 was carried out as a test of the general effectiveness of the refolding procedure.