PMID- 10585413
OWN - NLM
STAT- MEDLINE
DCOM- 20000113
LR  - 20190508
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 274
IP  - 50
DP  - 1999 Dec 10
TI  - Site-directed mutagenesis of diphosphoinositol polyphosphate phosphohydrolase, a 
      dual specificity NUDT enzyme that attacks diadenosine polyphosphates and
      diphosphoinositol polyphosphates.
PG  - 35434-40
AB  - 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.
FAU - Yang, X
AU  - Yang X
AD  - Inositide Signaling Group, Laboratory of Signal Transduction, NIEHS, National
      Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
      yang3@niehs.nih.gov
FAU - Safrany, S T
AU  - Safrany ST
FAU - Shears, S B
AU  - Shears SB
LA  - eng
PT  - Journal Article
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (DNA Primers)
RN  - 0 (Dinucleoside Phosphates)
RN  - 0 (Inositol Phosphates)
RN  - 0 (Recombinant Proteins)
RN  - 56983-23-4 (diadenosine 5',5''''-P1,P6-hexaphosphate)
RN  - EC 3.6.- (Acid Anhydride Hydrolases)
RN  - EC 3.6.1.- (diphosphoinositol polyphosphate phosphohydrolase)
SB  - IM
MH  - Acid Anhydride Hydrolases/*chemistry/isolation & purification/*metabolism
MH  - Amino Acid Sequence
MH  - Amino Acid Substitution
MH  - Circular Dichroism
MH  - DNA Primers
MH  - Dinucleoside Phosphates/*metabolism
MH  - Humans
MH  - Inositol Phosphates/*metabolism
MH  - Kinetics
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - Protein Conformation
MH  - Recombinant Proteins/chemistry/isolation & purification/metabolism
MH  - Saccharomyces cerevisiae/enzymology
MH  - Schizosaccharomyces/enzymology
MH  - Sequence Alignment
MH  - Sequence Homology, Amino Acid
MH  - Substrate Specificity
EDAT- 1999/12/10 00:00
MHDA- 1999/12/10 00:01
CRDT- 1999/12/10 00:00
PHST- 1999/12/10 00:00 [pubmed]
PHST- 1999/12/10 00:01 [medline]
PHST- 1999/12/10 00:00 [entrez]
AID - 10.1074/jbc.274.50.35434 [doi]
PST - ppublish
SO  - J Biol Chem. 1999 Dec 10;274(50):35434-40. doi: 10.1074/jbc.274.50.35434.