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Enzymatic hydrolysis by transition-metal-dependent nucleophilic aromatic substitution

Nature Chemical Biology volume 12, pages 1031–1036 (2016)Cite this article

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Abstract

Nitroaromatic compounds are typically toxic and resistant to degradation. Bradyrhizobium species strain JS329 metabolizes 5-nitroanthranilic acid (5NAA), which is a molecule secreted by Streptomyces scabies, the plant pathogen responsible for potato scab. The first biodegradation enzyme is 5NAA-aminohydrolase (5NAA-A), a metalloprotease family member that converts 5NAA to 5-nitrosalicylic acid. We characterized 5NAA-A biochemically and obtained snapshots of its mechanism. 5NAA-A, an octamer that can use several divalent transition metals for catalysis in vitro, employs a nucleophilic aromatic substitution mechanism. Unexpectedly, the metal in 5NAA-A is labile but is readily loaded in the presence of substrate. 5NAA-A is specific for 5NAA and cannot hydrolyze other tested derivatives, which are likewise poor inhibitors. The 5NAA-A structure and mechanism expand our understanding of the chemical ecology of an agriculturally important plant and pathogen, and will inform bioremediation and biocatalytic approaches to mitigate the environmental and ecological impact of nitroanilines and other challenging substrates.

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Figure 1: Overall structure and substrate binding features of 5NAA-A.
Figure 2: Dependence of 5NAA-A on metal ions for activity.
Figure 3: Metal ion, substrate, and product binding to 5NAA-A measured by ITC.
Figure 4: Metal coordination and proposed catalytic mechanism for 5NAA-A.

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Acknowledgements

This work was funded in part by a Pew Scholar and NSF CAREER award (0845445) to R.L.L., a Georgia Tech Molecular Biophysics Training Award to S.K., a Petit Undergraduate Research Fellowship and a President's Undergraduate Research Award to D.P.H., and a Georgia Internship for Teachers award to C.M.B. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. Work performed at Bio-CAT was supported by NIH NIGMS 9P41 GM103622. Use of the Pilatus 3 1M detector was provided by NIGMS 1S10OD018090-01.

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Author notes

  1. Sibel Kalyoncu and David P Heaner: These authors contributed equally to this work.

Authors and Affiliations

  1. School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, USA

    Sibel Kalyoncu, David P Heaner Jr, Casey M Bethel, Chiamaka U Ukachukwu & Raquel L Lieberman

  2. School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA

    Zohre Kurt & Jim C Spain

  3. Biophysics Collaborative Access Team, Advanced Photon Source, Argonne National Labs, Lemont, Illinois, USA

    Srinivas Chakravarthy

  4. Center for Environmental Diagnostics and Bioremediation, University of West Florida, Pensacola, Florida, USA

    Jim C Spain

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Contributions

S.K., R.L.L., D.P.H., and J.C.S. designed experiments. S.K., D.P.H., C.M.B., and Z.K. conducted enzymatic assays. S.K., D.P.H., and R.L.L. solved, refined, and analyzed crystal structures. C.M.B. and C.U.U. cloned, expressed, and purified enzymes. S.C. acquired and analyzed SAXS data. R.L.L., J.C.S., S.K., and D.P.H. wrote the manuscript.

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Correspondence to Raquel L Lieberman.

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Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–2 and Supplementary Figures 1–7. (PDF 8051 kb)

Supplementary Dataset 1

Phylogenetic tree of top 2255 protein sequences related to 5NAA-A. Red: 5NAA-A. (PDF 6829 kb)

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Kalyoncu, S., Heaner, D., Kurt, Z. et al. Enzymatic hydrolysis by transition-metal-dependent nucleophilic aromatic substitution. Nat Chem Biol 12, 1031–1036 (2016). https://doi.org/10.1038/nchembio.2191

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