| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
Nudix proteins are hydrolases that degrade toxic nucleotides and excess nucleotide derivatives in cells and thus called "house-cleaning enzymes. " Substrates of these enzymes are nucleoside diphosphate linked to other moiety, X. Nudes proteins are involved in maintenance of genetic information and regulation of cell proliferation and signal transduction by degrading their substrates. This study aimed to elucidate substrate-recognition and catalytic mechanisms of nudix proteins (Ndx1-8) from an extreme thermophile Thermus thermophilus HB8 at an atomic level, by structural and molecular functional analysis. First, the crystal structures of the substrate-bound complex of Ndx1, an Ap6A hydrolase, were determined. Mutational analysis based on the resolved structures revealed that adenine and three phosphate moieties are important for substrate recognition and that two glutamate residues coordinated to divalent rations in the active site. Second, we determined seven structures of Ndx4, ADP-ribose pyrophosphatas (ADPRase), including that of the free enzyme, the Zn^<2+>-bound enzyme, the binary complex with ADP-ribose, the ternary complexes with ADP-ribose and Zn^<2+> or Gd^<3+>, and the product complexes with AMP and Mg^<2+> or with ribose-5'-phosphate and Zn^<2+>. We have prepared mutants based on its crystal structure and analyzed their activity. We have identified residues involved in the substrate binding and reveal a unique mode of substrate recognition displayed by the ADPRase group of enzymes. Furthermore, mutational and kinetic studies of residues conserved in the nudix motif have allowed us to propose a new catalytic model. This model differs from that proposed for other nudix proteins. The crystal structure of Ndx2 was also determined. Our study demonstrates the diversity of molecular mechanisms shown in the nudix family of proteins.
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