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Site-directed Mutagenesis of Diphosphoinositol Polyphosphate Phosphohydrolase, a Dual Specificity NUDT Enzyme That Attacks Diadenosine Polyphosphates and Diphosphoinositol Polyphosphates*

Xiaonian Yang,S. Safrany,S. Shears

Published 1999 in Journal of Biological Chemistry

ABSTRACT

Diphosphoinositol polyphosphate phosphohydrolase (DIPP) hydrolyzes diadenosine 5′,5‴-P1,P6-hexaphosphate (Ap6A), a Nudix (nucleosidediphosphate attached-moiety “x”) substrate, and two non-Nudix compounds: diphosphoinositol pentakisphosphate (PP-InsP5) and bis-diphosphoinositol tetrakisphosphate ((PP)2-InsP4). Guided by multiple sequence alignments, we used site-directed mutagenesis to obtain new information concerning catalytically essential amino acid residues in DIPP. Mutagenesis of either of two conserved glutamate residues (Glu66 and Glu70) within the Nudt (Nudix-type) catalytic motif impaired hydrolysis of Ap6A, PP-InsP5, and (PP)2-InsP4 >95%; thus, all three substrates are hydrolyzed at the same active site. Two Gly-rich domains (glycine-rich regions 1 and 2 (GR1 and GR2)) flank the Nudt motif with potential sites for cation coordination and substrate binding. GR1 comprises a GGG tripeptide, while GR2 is identified as a new functional motif (GX 2GX 6G) that is conserved in yeast homologues of DIPP. Mutagenesis of any of these Gly residues in GR1 and GR2 reduced catalytic activity toward all three substrates by up to 95%. More distal to the Nudt motif, H91L and F84Y mutations substantially decreased the rate of Ap6A and (PP)2-InsP4 metabolism (by 71 and 96%), yet PP-InsP5 hydrolysis was only mildly reduced (by 30%); these results indicate substrate-specific roles for His91and Phe84. This new information helps define DIPP's structural, functional, and evolutionary relationships to Nudix hydrolases.

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