Bioremediation: Discovery of a Novel Alkaline Phosphatase through X-Ray Crystallography

Alkaline phosphatase (AP) is a bi-metalloenzyme with potential applications in biotechnology and bioremediation. The enzyme hydrolyses nonspecifically phosphate monoesters under alkaline conditions to yield inorganic phosphate. The AP isolated from the bacterium Sphingomonas sp. Strain BSAR-1 (SPAP) was shown to be particularly useful for the recovery of uranium from alkaline radioactive waste. We report here the first X-ray crystal structure of SPAP determined by using the Multi-wavelength Anomalous Diffraction method to solve the phase problem. The crystal structure shows that the enzyme active site is similar to those in other APs with two Zn ions in the core, even when there is a large difference in the rest of the tertiary structure. Structural differences observed are: 1) threonine as the catalytic residue instead of serine, 2) absence of third metal ion binding pocket, 3) deletion of the arginine residue forming bidentate hydrogen bonding with the substrate phosphoryl group, and 4) recruitment into the active site of lysine171 and aspargine110 residues to bind the substrate phosphoryl group in a manner not observed before in any other AP. These structural differences suggest that SPAP represents a new class of APs and also provides important insights into evolutionary relationships between members of AP superfamily. Because of its direct contact with the substrate phosphoryl group, lysine171 is proposed to play a significant role in catalysis.