Abstract Details

Functional and Structural Characterization of the Non-canonical Nucleotide Pyrophosphatase TM0159 from Thermotoga maritima
Abstract ID	BIO-05
Presenter	Khaldeyah  Awwad
Presentation Type	Poster
Full Author List	K. Awwad (1) , A. Desai (1) , W. Waitkus-Roberts (1) , S. Huang (1) , K. Pham (1) , M. Sommerhalter (1)
Affiliations	(1) CSU East Bay, Hayward CA, USA
Category	Bio/Life Sciences
Abstract	The structure of TM0159 from Thermotoga maritima was solved by the Joint Center for Structural Genomics (JCSG).(1) Based on a high structural similarity to other proteins TM0159 most likely belongs to a novel class of non-canonical pyrophosphatases.(2-5) These enzymes exhibit an important “house-cleaning” function by removing erroneous nucleotide triphosphates from the precursor pool of RNA and DNA building blocks. We confirmed the suggested functional role of TM0159 by using different enzymatic assay methods, including thin layer chromatography, high-performance liquid chromatography, and colorimetric assays. TM0159 is capable to convert erroneous (non-canonical) nucleotides such as inosine triphosphate (ITP) and xanthine triphosphate (XTP) into their corresponding monophosphate nucleotides (IMP and XMP). Canonical nucleotides such as adenosine triphosphate (ATP) or guanosine triphosphate (GTP) are not converted to any significant extent. Our next goal is to elucidate how TM0159 is capable to discriminate between non-canonical and canonical nucleotides by solving the X-ray structure of TM0159 in the presence of different nucleotides. We prepared crystals of apo-TM0159 and soaked them in a solution containing the nucleotide analogue APcPP (adenosine-5’-(alpha,beta)-methyleno-triphosphate). X-ray diffraction experiments at SSRL BL 9-1 revealed extensive crystal damage. As a result of the soaking procedure all investigated crystals exhibited very poor diffraction quality. Next, we preincubated TM0159 with different nucleotides (APcPP, ITP, IMP, XTP, and XMP) and screened for new crystallization conditions. In the absence of Mg2+ or Mn2+ neither ITP nor XTP are cleaved by TM0159. We obtained several hits with different precipitants, including malic acid, polyethylene glycol 8000 (PEG8000), PEG3350, PEG-MME500, jeffamine M-600, pentaerythritolpropoxylate, and methanepentanediol (MPD). Currently, we are optimizing these new crystallization conditions to perform new X-ray diffraction experiments at SSRL.
Footnotes	1. Joint Center for Structural Genomics. Crystal structure of putative Xanthosine triphosphate pyrophosphatase/HAM1 protein homolog (TM0159) from Thermotoga maritima at 1.78 A resolution, 2004, to be published (PDB-ID: 1vp2). 2. K.Y. Hwang, J.H. Chung, S.H. Kim, Y.S. Han, and Y. Cho. Structure-based identification of a novel NTPase from Methanococcus jannaschii. Nature Structural Biology 6:691-696 (1999). 3. A. Savchenko, M. Proudfoot, T. Skarina, A. Singer, O. Litvinova, R. Sanishvili, G. Brown, N. Chirgadze, and A.F. Yakunin. Molecular basis of the antimutagenic activity of the house-cleaning inosine triphosphate pyrophosphatase RdgB from Escherichia coli. Journal of Molecular Biology 374:1091-1103 (2007). 4. N.K. Lokanath, K.J. Pampa, K. Takio, and N. Kunishima. Structures of dimeric nonstandard nucleotide triphosphate pyrophosphatase from Pyrococcus horikoshii OT3: functional significance of interprotomer conformational changes. Journal of Molecular Biology 375:1013-1025 (2008). 5. P. Stenmark, P. Kursula, S. Flodin, S. Graslund, R. Landry, P. Nordlund, and H. Schuler. Crystal structure of human inosine triphosphatase. Substrate binding and implication of the inosine triphosphatase deficiency mutation P32T. The Journal of Biological Chemistry 282:3182-3187 (2007).
Funding Acknowledgement	We would like to thank SSRL, their friendly staff, and Prof. Dr. K. Kantardjieff (CSU Fullerton & CMolS) for facilitating beam-time at SSRL. Maggie Kit-Man Ho, Chieu Phan and Vu Tran assisted during the early stages of protein purification. Funding for this project was obtained from the CSU Faculty-Student Collaborative Research Seed Grant Program (CSUPERB) and a Faculty Support Grant (CSU East Bay). A. Desai received a Graduate Student Research Grant (CSU East Bay).