Structural elucidation of a dual-activity PAP phosphatase-1 fromEntamoeba histolyticacapable of hydrolysing both 3′-phosphoadenosine 5′-phosphate and inositol 1,4-bisphosphate

Abstract

The enzyme 3′-phosphoadenosine 5′-phosphatase-1 (PAP phosphatase-1) is a member of the Li+-sensitive Mg2+-dependent phosphatase superfamily, or inositol monophosphatase (IMPase) superfamily, and is an important regulator of the sulfate-activation pathway in all living organisms. Inhibition of this enzyme leads to accumulation of the toxic byproduct 3′-phosphoadenosine 5′-phosphate (PAP), which could be lethal to the organism. Genomic analysis ofEntamoeba histolyticasuggests the presence of two isoforms of PAP phosphatase. The PAP phosphatase-1 isoform of this organism is shown to be active over wide ranges of pH and temperature. Interestingly, this enzyme is inhibited by submillimolar concentrations of Li+, while being insensitive to Na+. Interestingly, the enzyme showed activity towards both PAP and inositol 1,4-bisphosphate and behaved as an inositol polyphosphate 1-phosphatase. Crystal structures of this enzyme in its native form and in complex with adenosine 5′-monophosphate have been determined to 2.1 and 2.6 Å resolution, respectively. The PAP phosphatase-1 structure is divided into two domains, namely α+β and α/β, and the substrate and metal ions bind between them. This is a first structure of any PAP phosphatase to be determined from a human parasitic protozoan. This enzyme appears to function using a mechanism involving three-metal-ion assisted catalysis. Comparison with other structures indicates that the sensitivity to alkali-metal ions may depend on the orientation of a specific catalytic loop.