Structural and Functional Analysis of Phospholipid‐Specific Nucleotide Pyrophosphatase/ Phosphodiesterase Isoforms

Abstract

Lysophosphatidic acid (LPA) is an important mediator in development and disease. Despite roles in human health, little is known about its biosynthesis and regulation. In 2002, autotaxin (NPP2) was shown to produce LPA in plasma. We are using computational, biochemical, and biophysical approaches to examine the structure and function of NPP2/6/7. Previous work suggested that these three isoforms show differences in regioselectivity for hydrolysis of lysophosphatidyl choline (LPC). Likewise, these isoforms are differentially sensitive to the removal and subsequent replacement of divalent cations. Recombinant proteins were expressed in insect cells and were purified by affinity chromatography. The isoforms showed similar ability to utilize LPC as substrate, but different abilities to hydrolyze other substrates. EDTA inhibited all three isoforms with maximal effect at 16h and 120‐fold excess chelator. Following EDTA treatment, Zn2+ was able to restore activity to all three isoforms, whereas Co2+ differentially restored activity. Significant effects on secondary structure or thermal stability was not seen for either treatment (CD). Homology models based on the Xac NPP crystal structure (2gsu) have been used to guide site‐directed mutagenesis. Mutation of the reported catalytic residues to alanine, gives catalytically dead proteins. Characterization of additional mutations is ongoing.