Structural Biology of the Nudix Proteins from the Extremely Radiation-Resistant Bacterium Deinococcus radiodurans
EMSL Project ID
2331a
Abstract
The bacterium Deinococcus radiodurans is able to withstand high doses of ionizing and UV radiation that are lethal to virtually all other organisms (Battista, 1997). It has been suggested that this resistance to radiation and to other DNA-damaging agents (including hydrogen peroxide and mitomycin C) may be due to unusually efficient DNA repair mechanisms (Minton, 1994). Consequently, D. radiodurans is one of the leading candidates for the bioremediation of radioactive waste and a subject for the study of DNA repair mechanisms.The complete genome of D. radiodurans has recently been sequenced (White et al., 1999). Analysis of this sequence reveals a full suite of genes with potential DNA repair activities, essentially all of which have functional homologues in other procaryotes. Interestingly, these hypothetical DNA repair genes exhibit a high amount of redundancy that includes 23 genes that have sequence homology with the Nudix family of polyphosphate pyrophosphohydrolases. Nudix proteins are found in all organisms and are identified by the consensus sequence GX5EX7REUXEEXGU (where U = I, L, or V and X = any amino acid) that forms part of the catalytic site for the hydrolysis of a NUcleoside DIphosphate linked to some other moiety, X. The prototypical member of this family is the Escherichia coli MutT protein that catalyzes hydrolysis of nucleotide triphosphates with a preference for 8-oxo-dGTP. The primary function of Nudix proteins are to sanitize the cell by reducing the level of potentially mutagenic and/or toxic compounds and the accumulation of biochemical intermediates. While more than 450 puntative Nudix proteins have been identified in genomes on the basis of the Nudix consensus sequence, few Nudix protein structures have been determined. For those few structures that have been determined, beyond the conserved cataylic core there is a considerable variation in the peripheral structure and oligomerization state. To better understand the relevance, function, and mechanisms of the Nudix family of proteins, and to better understand the roles played by the hypothetical D. radiodurans Nudix proteins in radiation-resistance, we have targeted the 23 hypothetical Nudix proteins for structure determination. Using the NMR facilities at the Environmental Molecular Sciences Laboratory we have collected data that allowed us to complete the solution structure for the hypothetical D. radiodurans Nudix protein DR0079, a 171 residue, 19.3 kDa protein (Buchko et al., in preparation). Our request for additional NMR time is to (i) refine the DR0079 structure in the presence of Mg2+, a divalent metal required for enzymatic activity, (ii) determine if the substrate for DR0079 are oxidized NTPs, and (iii) screen other hypothetical D. radiodurans Nudix proteins for structure determination via NMR methods.
Project Details
Project type
Capability Research
Start Date
2003-10-01
End Date
2004-04-09
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Buchko, GW (2010) "Circular dichroism studies on the Deinococcus radiodurans Nudix hydrolase DR_0079: An atypical thermal melt." Protein and Peptide Letters. 17:831-835.
Buchko GW, O Litvinova, H Robinson, AF Yakunin, and MA Kennedy. 2008. "Functional and structural characterization of DR_0079 from Deinococcus radiodurans, a novel Nudix hydrolase with a preference for cytosine (deoxy) ribonucleoside 5'-di- and triphosphates." Biochemistry 47(25):6571-6582.
Buchko GW, S Ni, SR Holbrook, and MA Kennedy. 2003. "H-1, C-13, and N-15 NMR assignments of the hypothetical Nudix protein DR0079 from the extremely radiation-resistant bacterium Deinococcus radiodurans.." Journal of Biomolecular NMR 25(2):169-170.
Buchko GW, S Ni, SR Holbrook, and MA Kennedy. 2004. "Solution Structure of Hypothetical Nudix Hydrolase DR0079 from Extremely Radiation-Resistant Deinococcus radiodurans Bacterium." Proteins. Structure, Function, and Bioinformatics 56(1):28-39.
E.L. Holbrook, U. Schulze-Gahmen, G.W. Buchko, S. Ni, M.A. Kennedy, and S.R. Holbrook. (2003) Purification, crystallization and preliminary X-ray analysis of two Nudix hydrolases from Deinococcus radiodurans. Acta Crystallogr. D Biol. Crystallogr., 59:737-740.