Site-directed mutagenesis of diphosphoinositol polyphosphate phosphohydrolase, a dual specificity NUDT enzyme that attacks diadenosine polyphosphatesand diphosphoinositol polyphosphates

Diphosphoinositol polyphosphate phosphohydrolase (DIPP) hydrolyzes diadenos ine 5',5"-P-1,P-6-hexaphosphate (Ap(6)A), a Nudix (nucleoside diphosphate a ttached-moiety "x") substrate, and two non-Nudix compounds: diphosphoinosit ol pentakisphosphate (PP-InsP(5),) and bis-diphosphoinositol tetrakisphosph ate (PP)(2)-InsP(4)), Guided by multiple sequence alignments, we used site- directed mutagenesis to obtain new information concerning catalytically ess ential amino acid residues in DIPP, Mutagenesis of either of two conserved glutamate residues (Glu(66) and Glu(70)) within the Nudt (Nudix-type) catal ytic motif impaired hydrolysis of Ap(6)A, PP-InsP(5), and (PP)(2)-InsP(4) > 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 Nud t motif with potential sites for cation coordination and substrate binding. GR1 comprises a GGG tripeptide, while GR2 is identified as a new functiona l motif (GX(2)GX(6)G) that is conserved in yeast homologues of DIPP. Mutage nesis of any of these Gly residues in GR1 and GR2 reduced catalytic activit y toward all three substrates by up to 95%. More distal to the Nudt motif, H91L and F84Y mutations substantially decreased the rate of Ap(6)A and (PP) (2)-InsP(4) metabolism (by 71 and 96%), yet PP-InsP(5) hydrolysis was only mildly reduced (by 30%); these results indicate substrate-specific roles fo r His(91) and phe(84), This new information helps define DIPP's structural, functional, and evolutionary relationships to Nudix hydrolases.