X-ray structure reveals a new class and provides insight into evolution of alkaline phosphatases.

The alkaline phosphatase (AP) is a bi-metalloenzyme of potential applications in biotechnology and bioremediation, in which phosphate monoesters are nonspecifically hydrolysed under alkaline conditions to yield inorganic phosphate. The hydrolysis occurs through an enzyme intermediate in which the catalytic residue is phosphorylated. The reaction, which also requires a third metal ion, is proposed to proceed through a mechanism of in-line displacement involving a trigonal bipyramidal transition state. Stabilizing the transition state by bidentate hydrogen bonding has been suggested to be the reason for conservation of an arginine residue in the active site. We report here the first crystal structure of alkaline phosphatase purified from the bacterium Sphingomonas. sp. Strain BSAR-1 (SPAP). The crystal structure reveals many differences from other APs: 1) the catalytic residue is a threonine instead of serine, 2) there is no third metal ion binding pocket, and 3) the arginine residue forming bidentate hydrogen bonding is deleted in SPAP. A lysine and an aspargine residue, recruited together for the first time into the active site, bind the substrate phosphoryl group in a manner not observed before in any other AP. These and other structural features suggest that SPAP represents a new class of APs. Because of its direct contact with the substrate phosphoryl group, the lysine residue is proposed to play a significant role in catalysis. The structure is consistent with a mechanism of in-line displacement via a trigonal bipyramidal transition state. The structure provides important insights into evolutionary relationships between members of AP superfamily

Crystallization and preliminary X-ray diffraction analysis of human seminal plasma protein PSP94

The human seminal plasma protein PSP94 has been purified from human seminal plasma and crystallized

Refinement statistics for native SPAP.

<p>*The numbers between parentheses indicate the value in the outer resolution shell. R.m.s =  root mean square.</p

Novel hydrogen bonding interactions in SPAP: (A) Hydrogen bonding network involving His93 (B) Conservation of hydrogen bond from Asp300 OD1 through correlated sequence differences in SPAP (pink) and ECAP (slate).

<p>Distances are in Å unit.</p

Size Exclusion Chromatography Elution profile of SPAP.

<p>Standards used are shown in red along with their molecular weights. SPAP, shown in blue, elutes as a monomer of molecular weight 58 kD.</p

Unique disulfide bond near the active site: unique disulfide bond Cys90 – Cys126 next to the catalytic residue Thr89 may be crucial for the integrity of active site geometry.

<p>Unique disulfide bond near the active site: unique disulfide bond Cys90 – Cys126 next to the catalytic residue Thr89 may be crucial for the integrity of active site geometry.</p

Data collections and phasing statistics for SM-SPAP.

<p>The numbers between parentheses indicate the value in the outer resolution shell.</p

Relative orientation of hydroxyl group of catalytic residues at different stages: structural superposition in present structure (magenta), phosphoenzyme intermediate (green) and product complex (light blue).

<p>Note that Thr89 hydroxyl overlaps the ester oxygen in the phosphoenzyme complex.</p