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

• Published in: The Journal of biological chemistry Vol. 274; no. 50; pp. 35434 - 35440
• Main Authors: Yang, X, Safrany, S T, Shears, S B
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 10.12.1999
• Subjects: Acid Anhydride Hydrolases - chemistry; Acid Anhydride Hydrolases - isolation & purification; Acid Anhydride Hydrolases - metabolism; Amino Acid Sequence; Amino Acid Substitution; Circular Dichroism; diadenosine polyphosphates; Dinucleoside Phosphates - metabolism; Diphosphoinositol polyphosphate phosphohydrolase; diphosphoinositol polyphosphates; DNA Primers; Humans; Inositol Phosphates - metabolism; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - enzymology; Schizosaccharomyces - enzymology; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity
• DOI: 10.1074/jbc.274.50.35434
• ISSN: 0021-9258

Diphosphoinositol polyphosphate phosphohydrolase (DIPP) hydrolyzes diadenosine 5′,5‴-P 1 ,P 6 -hexaphosphate (Ap 6 A), a Nudix ( n ucleoside di phosphate 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 ( N udix- t ype) 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 (G X 2 G X 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.