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Discovery of a Ni2+-dependent guanidine hydrolase in bacteria

Nature volume 603, pages 515–521 (2022)Cite this article

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Abstract

Nitrogen availability is a growth-limiting factor in many habitats1, and the global nitrogen cycle involves prokaryotes and eukaryotes competing for this precious resource. Only some bacteria and archaea can fix elementary nitrogen; all other organisms depend on the assimilation of mineral or organic nitrogen. The nitrogen-rich compound guanidine occurs widely in nature2,3,4, but its utilization is impeded by pronounced resonance stabilization5, and enzymes catalysing hydrolysis of free guanidine have not been identified. Here we describe the arginase family protein GdmH (Sll1077) from Synechocystis sp. PCC 6803 as a Ni2+-dependent guanidine hydrolase. GdmH is highly specific for free guanidine. Its activity depends on two accessory proteins that load Ni2+ instead of the typical Mn2+ ions into the active site. Crystal structures of GdmH show coordination of the dinuclear metal cluster in a geometry typical for arginase family enzymes and allow modelling of the bound substrate. A unique amino-terminal extension and a tryptophan residue narrow the substrate-binding pocket and identify homologous proteins in further cyanobacteria, several other bacterial taxa and heterokont algae as probable guanidine hydrolases. This broad distribution suggests notable ecological relevance of guanidine hydrolysis in aquatic habitats.

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Fig. 1: GdmH (Sll1077) is a Ni2+-dependent guanidine hydrolase.
Fig. 2: Kinetic and energetic properties of GdmH.
Fig. 3: Structural characterization of GdmH.
Fig. 4: Growth of Synechocystis sp. PCC 6803 with guanidine as the sole source of nitrogen.

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Data availability

Structure coordinates of GdmH from Synechocystis sp. PCC6803 and experimental structure factor amplitudes have been deposited with the Protein Data Bank as entries 7OI1 and 7ESR, for space groups C2 and R32, respectively. X-ray diffraction images have been deposited with Zenodo, at which https://doi.org/10.5281/zenodo.4750963 corresponds to PDB entry 7OI1 and https://doi.org/10.5281/zenodo.4750940corresponds to 7ESR. The raw data are presented in the Article and are available from the corresponding authors upon reasonable request.

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Acknowledgements

J.S.H. acknowledges the ERC CoG project 681777 “RiboDisc” for financial support. R.L.-I. was supported by grant PID2019-104784RJ-100 MCIN/AEI/10.13039/501100011033 Spain, and grant BFU2017-88202-P MCIN/AEI/10.13039/501100011033 Spain, cofinanced by ERDF A way of making Europe. We thank R. Winter and E. Flores for helpful discussions and A. Joachimi and D. Galetskiy for technical assistance. We acknowledge the Paul Scherrer Institut, Villigen, Switzerland, for provision of synchrotron radiation beamtime at beamline X06SA-PXI of the Swiss Light Source, and thank K. M. L. Smith for assistance. We thank K. Forchhammer for providing the Synechocystis PCC 6803 GT cells and for helpful advice regarding their cultivation.

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Author notes

  1. These authors contributed equally: D. Funck, M. Sinn

Authors and Affiliations

  1. Department of Chemistry, University of Konstanz, Konstanz, Germany

    D. Funck, M. Sinn, M. Stanoppi, J. Dietrich & J. S. Hartig

  2. Department of Biology, University of Konstanz, Konstanz, Germany

    J. R. Fleming & O. Mayans

  3. Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla and C.S.I.C, Seville, Spain

    R. López-Igual

  4. Konstanz Graduate School Chemical Biology (KoRS-CB), University of Konstanz, Konstanz, Germany

    O. Mayans & J. S. Hartig

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Contributions

D.F., M. Sinn and J.S.H. conceived the project. D.F. and M. Sinn performed protein expression, purification, activity assays and sequence analyses. R.L.-I. generated the Δsll1077 mutant and J.D., R.L.-I. and D.F. performed growth assays. M. Stanoppi performed NMR analyses. J.R.F. and O.M. performed protein crystallization, structure determination, analysis and modelling. D.F., M. Sinn and J.S.H. wrote the manuscript with input from all authors. D.F., M. Sinn, J.D., M. Stanoppi and J.R.F. prepared figures. The manuscript was reviewed and approved by all coauthors.

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Correspondence to J. S. Hartig.

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Extended data figures and tables

Extended Data Fig. 1 Additional characterization GdmH.

a: Activation of GdmH expressed in the absence of added metal ions and either co-expressed with GhaA and GhaB or with either of the accessory proteins alone by Ni2+ or Fe2+. The orange columns represent the activity in a mixture of the latter two extracts. Where indicated, 1 mM β-mercaptoethanol (β-MSH) was additionally included. Columns represent the average of n = 3 technical replicates, error bars indicate s.d. The entire experiment was repeated with independent bacterial extracts, which yielded slightly different absolute values for specific activities but virtually identical results for the relative values. b: 13C-NMR spectra of 13C15N-guanidine after incubation over night with purified GdmH (overexpressed together with GhaA and GhaB). Exclusively the triplet signal of 13C15N-urea was detected after incubation with purified GdmH, whereas after incubation with GdmH partially inactivated by heat treatment, both the quadruplet of 13C15N-guanidine and the triplet signal of 13C15N-urea were detected. c: Coupled enzymatic assay of GdmH with glutamate dehydrogenase (GDH). NADH gets oxidized by GDH during reductive amination of α-ketoglutarate with ammonia released from guanidine by GdmH. Michaelis-constant KM, maximal specific activity Amax, and catalytic constant kcat were determined using different guanidine concentrations. Data points represent the average of n = 3 technical replicates and the black line represents the least-square fit to the Michaelis-Menten equation. Error bars represent s.d. The whole experiment was repeated with an independent enzyme preparation, which gave consistent results. d: Influence of Na-cacodylate on the activity of GdmH with 10 mM guanidine as substrate. A cacodylate molecule occupied the active site in one of the crystal structures but even a tenfold excess of cacodylate had no influence on GdmH activity. Columns represent the average of n = 3 independent enzyme preparations, error bars indicate s.d. e: Effect of point mutations on the activity and KM of purified, recombinant GdmH. The black lines represent the least square fits to the Michaelis-Menten equation of single experiments. The entire analysis was repeated with independent enzyme preparations with consistent results. The inset shows a Coomassie-stained gel with the purified GdmH variants and the positions of molecular weight markers. For gel source data, see Supplementary Fig. 1b. f: Time-dependence of urea production by GdmH in the presence of 10 mM guanidine. Note that almost half of the substrate was hydrolyzed after 3 days of incubation.

Extended Data Fig. 2 Additional images of the GdmH structure.

a: Top view of the GdmH hexamer with one subunit in surface display (bright yellow) and two subunits as ribbons below a transparent surface (orange and sky blue). The extended N-terminus of the orange subunit is highlighted by saturated color. b: Comparison of the GdmH capping helix (blue) with the same helix from the most similar protein with a high resolution structure, guanidinobutyrase from Pseudomonas aeruginosa (grey with capping helix in yellow, PDB entry 3NIO). Although the overall fold is well conserved between both enzymes, the position of the highlighted α-helix is shifted towards the active site.

Extended Data Fig. 3 Relation of GdmH to other proteins from the arginase superfamily.

Unrooted neighbor-joining tree of 509 representatives of >15000 UniRef90 sequences with at least 27% identity to residues 60–390 of GdmH, selected and aligned with the ConSurf webserver61. Accession numbers of all sequences are given in Supplementary Data 2. The branches are colored according to the taxonomic group: Black: bacteria; cyan: archaea; bright green: fungi; green: plants; brown: stramenopile; magenta: animals. Branches containing selected enzymes with biochemically confirmed function are highlighted and demonstrate that very dissimilar sequences can have the same enzymatic activity in different taxa.

Extended Data Table 1 Kinetic properties of Synechocystis guanidine hydrolase GdmH
Full size table
Extended Data Table 2 X-ray diffraction data collection and refinement statistics
Full size table

Supplementary information

Supplementary Fig. 1

Source data for SDS gel (Fig. 1e); source data for SDS gel (Extended Data Fig. 1e) as pdf file.

Reporting Summary

Peer Review File

Supplementary Data 1

Multiple-sequence alignment of GdmH with similar sequences as fasta formatted plain text file.

Supplementary Data 2

Manually optimized sequence alignment of GdmH with further sequences of characterized enzymes of the arginase family (source data for Extended Data Fig. 3) as fasta formatted plain text file.

Supplementary Data 3

Multiple-sequence alignment of the 194 closest homologues of GdmH (source data for Fig. 3g) as fasta formatted plain text file.

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Funck, D., Sinn, M., Fleming, J.R. et al. Discovery of a Ni2+-dependent guanidine hydrolase in bacteria. Nature 603, 515–521 (2022). https://doi.org/10.1038/s41586-022-04490-x

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