A nuclear magnetic resonance based approach to accurate functional annotation of putative enzymes in the methanogen Methanosarcina acetivorans

BMC Genomics

Research

Libuse Brachova¹  Basil J Nikolau²  Kevin Sowers³  Ethel Apolinario³  Zhuo Li⁴  Yihong Chen⁴  Zvi Kelman⁵  John Orban⁶  Lucas Showman⁷ 

[1] Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, 50011, Ames, IA, USA;Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, 50011, Ames, IA, USA;Center for Biorenewable Chemicals, Iowa State University, 50011, Ames, IA, USA;Plant Sciences Institute, Iowa State University, 50011, Ames, IA, USA;Department of Marine Biotechnology, University of Maryland Baltimore County, Baltimore, MD, USA;Institute for Bioscience and Biotechnology Research, University of Maryland, 9600 Gudelsky Drive, 20850, Rockville, MD, USA;Institute for Bioscience and Biotechnology Research, University of Maryland, 9600 Gudelsky Drive, 20850, Rockville, MD, USA;Department of Cell Biology and Molecular Genetics, University of Maryland College Park, USA;Institute for Bioscience and Biotechnology Research, University of Maryland, 9600 Gudelsky Drive, 20850, Rockville, MD, USA;Department of Chemistry and Biochemistry, University of Maryland College Park, USA;Plant Sciences Institute, Iowa State University, 50011, Ames, IA, USA;

关键词: Nuclear Magnetic Resonance;    Nuclear Magnetic Resonance Experiment;    Functional Assignment;    Nuclear Magnetic Resonance Sample;    Putative Enzyme;   

DOI  :  10.1186/1471-2164-12-S1-S7

来源: Springer

【 摘 要 】

BackgroundCorrect annotation of function is essential if one is to take full advantage of the vast amounts of genomic sequence data. The accuracy of sequence-based functional annotations is often variable, particularly if the sequence homology to a known function is low. Indeed recent work has shown that even proteins with very high sequence identity can have different folds and functions, and therefore caution is needed in assigning functions by sequence homology in the absence of experimental validation. Experimental methods are therefore needed to efficiently evaluate annotations in a way that complements current high throughput technologies. Here, we describe the use of nuclear magnetic resonance (NMR)-based ligand screening as a tool for testing functional assignments of putative enzymes that may be of variable reliability.ResultsThe target genes for this study are putative enzymes from the methanogenic archaeon Methanosarcina acetivorans (MA) that have been selected after manual genome re-annotation and demonstrate detectable in vivo expression at the level of the transcriptome. The experimental approach begins with heterologous E. coli expression and purification of individual MA gene products. An NMR-based ligand screen of the purified protein then identifies possible substrates or products from a library of candidate compounds chosen from the putative pathway and other related pathways. These data are used to determine if the current sequence-based annotation is likely to be correct. For a number of case studies, additional experiments (such as in vivo genetic complementation) were performed to determine function so that the reliability of the NMR screen could be independently assessed.ConclusionsIn all examples studied, the NMR screen was indicative of whether the functional annotation was correct. Thus, the case studies described demonstrate that NMR-based ligand screening is an effective and rapid tool for confirming or negating the annotated gene function of putative enzymes. In particular, no protein-specific assay needs to be developed, which makes the approach broadly applicable for validating putative functions using an automated pipeline strategy.

【 授权许可】

Unknown   
© Chen et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.