Site-directed mutagenesis of diphosphoinositol polyphosphate phosphohydrolase, a dual specificity NUDT enzyme that attacks diadenosine polyphosphates and diphosphoinositol polyphosphates.
Diphosphoinositol polyphosphate phosphohydrolase (DIPP) hydrolyzes diadenosine 5',5"'-P(1),P(6)-hexaphosphate (Ap(6)A), a Nudix (nucleoside diphosphate attached-moiety "x") substrate, and two non-Nudix compounds: diphosphoinositol pentakisphosphate (PP-InsP(5)) and bis-diphosphoinositol tetrakisphosphate ((PP)(2)-InsP(4)). 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 (Glu(66) and Glu(70)) within the Nudt (Nudix-type) catalytic 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 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(2)GX(6)G) 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 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 for His(91) and Phe(84). This new information helps define DIPP's structural, functional, and evolutionary relationships to Nudix hydrolases.
Yang
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Inositide Signaling Group, Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA. yang3@niehs.nih.gov
Safrany
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Shears
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D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D017931:DNA Primers; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012568:Schizosaccharomyces / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D015226:Dinucleoside Phosphates; D007295:Inositol Phosphates; D011994:Recombinant Proteins; C070070:diadenosine 5',5''''-P1,P6-hexaphosphate; D017766:Acid Anhydride Hydrolases; C116368:diphosphoinositol polyphosphate phosphohydrolase