Altering of the Metal Specificity of Escherichia coliAlkaline Phosphatase

Abstract

Analysis of sequence alignments of alkaline phosphatases revealed a correlation between metal specificity and certain amino acid side chains in the active site that are metal-binding ligands. The Zn 2؉ -requiring Escherichia coli alkaline phosphatase has an Asp at position 153 and a Lys at position 328. Co 2؉ -requiring alkaline phosphatases from Thermotoga maritima and Bacillus subtilis have a His and a Trp at these positions, respectively. The mutations D153H, K328W, and D153H/ K328W were induced in E. coli alkaline phosphatase to determine whether these residues dictate the metal dependence of the enzyme. The wild-type and D153H enzymes showed very little activity in the presence of Co 2؉ , but the K328W and especially the D153H/K328W enzymes effectively use Co 2؉ for catalysis. Isothermal titration calorimetry experiments showed that in all cases except for the D153H/K328W enzyme, a possible conformation change occurs upon binding Co 2؉ . These data together indicate that the active site of the D153H/ K328W enzyme has been altered significantly enough to allow the enzyme to utilize Co 2؉ for catalysis. These studies suggest that the active site residues His and Trp at the E. coli enzyme positions 153 and 328, respectively, at least partially dictate the metal specificity of alkaline phosphatase.

Characterization of a monomeric heat-labile classical alkaline phosphatase from Anabaena sp. PCC7120

“…This difference might have resulted from changes in the amino acids required for ion binding. Similar to the EAP double mutant D153H/K328W, which is also activated by Co instead of Zn [34], the cor responding residues in AP A are histidine and tryptophan, exactly the same as in the EAP mutant D153H/K328W.…”

Coordination sphere of the third metal site is essential to the activity and metal selectivity of alkaline phosphatases

“…2). This binding site is well suited to octahedral 25 However, the coordination network of active sites of these variants differs from the TAP active site. ECAP 153H adjusts to directly coordinate M3, unlike in TAP, which preserves the residue 153 coordination and coordination through a water molecule.…”

Gene Cloning, Overproduction, and Characterization of Thermolabile Alkaline Phosphatase from a Psychrotrophic Bacterium

“…E. coli AC109-(DE3) was transformed with pET-SAP, in which the transcription of the SIB1 APase gene is controlled by the T7 promoter, and grown at 37 C. When the absorbance at 660 nm of the culture reached 0.5-1.0, 1 mM of isopropyl -D-thiogalactopyranoside (IPTG) was added to the culture medium and cultivation was continued at 15 C for 15-20 h. Cells were then harvested by centrifugation at 4000 g for 7 min. Recombinant proteins localized in the periplasmic space of the E. coli cells were released into the external medium by osmotic shock 26) and fractionated by ammonium sulfate precipitation.…”

“…9) Of these APases, E. coli APase has been the most extensively studied for structures and functions. [10][11][12][13][14][15][16] This enzyme is located in the periplasmic space as a homodimer and is involved in the acquisition of phosphate from esters when free inorganic phosphate is depleted. Each subunit is composed of 449 amino acid residues and contains two Zn 2þ and one Mg 2þ ions.…”