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The pimeloyl-CoA synthetase BioW defines a new fold for adenylate-forming enzymes

Nature Chemical Biology volume 13, pages 668–674 (2017)Cite this article

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Abstract

Reactions that activate carboxylates through acyl-adenylate intermediates are found throughout biology and include acyl- and aryl-CoA synthetases and tRNA synthetases. Here we describe the characterization of Aquifex aeolicus BioW, which represents a new protein fold within the superfamily of adenylating enzymes. Substrate-bound structures identified the enzyme active site and elucidated the mechanistic strategy for conjugating CoA to the seven-carbon α,ω-dicarboxylate pimelate, a biotin precursor. Proper position of reactive groups for the two half-reactions is achieved solely through movements of active site residues, as confirmed by site-directed mutational analysis. The ability of BioW to hydrolyze adenylates of noncognate substrates is reminiscent of pre-transfer proofreading observed in some tRNA synthetases, and we show that this activity can be abolished by mutation of a single residue. These studies illustrate how BioW can carry out three different biologically prevalent chemical reactions (adenylation, thioesterification, and proofreading) in the context of a new protein fold.

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Figure 1: Reaction catalyzed by BioW and representative homologs.
Figure 2: Biochemical activity of AaBioW.
Figure 3: Crystal structures of AaBioW and ligand complexes.
Figure 4: Structure-based mutational analysis of the AaBioW active site.
Figure 5: Proofreading activity of the wild-type and R159A AaBioWs.

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Acknowledgements

We thank the Mining Microbial Genomes theme at the Carl R. Woese Institute for Genomic Biology for access to the LC–MS equipment. We also thank K. Brister and the staff at the Life Sciences Collaborative Access Team (Sector 21) at the Argonne National Labs for facilitating data collection. This work was supported by NIH grant AI15650 (to J.C.).

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Authors and Affiliations

  1. Department of Biochemistry, University of Illinois at Urbana–Champaign, Urbana, Illinois, USA

    Paola Estrada, Shi-Hui Dong, John E Cronan & Satish K Nair

  2. Department of Microbiology, University of Illinois at Urbana–Champaign, Urbana, Illinois, USA

    Miglena Manandhar & John E Cronan

  3. Institute for Genomic Biology University of Illinois at Urbana–Champaign, Urbana, Illinois, USA

    Shi-Hui Dong, Jaigeeth Deveryshetty, Vinayak Agarwal, John E Cronan & Satish K Nair

  4. Center for Biophysics and Computational Biology, University of Illinois at Urbana–Champaign, Urbana, Illinois, USA

    Vinayak Agarwal & Satish K Nair

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  1. Paola Estrada
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Contributions

S.K.N. and J.E.C. conceived the studies. P.E. designed and performed most of the crystallographic and biochemical experiments with the following exceptions: V.A. carried out crystallization of BaBioW, J.D. performed crystallization of SeMet AaBioW, and M.M. carried out the thin-layer chromatographic analysis. S.-H.D. assisted in mass spectrometric analysis. S.K.N. wrote the manuscript with input from P.E., M.M., and J.E.C.

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Correspondence to Satish K Nair.

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Estrada, P., Manandhar, M., Dong, SH. et al. The pimeloyl-CoA synthetase BioW defines a new fold for adenylate-forming enzymes. Nat Chem Biol 13, 668–674 (2017). https://doi.org/10.1038/nchembio.2359

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