Abstract
Many bacteriophages evade bacterial immune recognition by substituting adenine with 2,6-diaminopurine (Z) in their genomes. The Z-genome biosynthetic pathway involves PurZ that belongs to the PurA (adenylosuccinate synthetase) family and bears particular similarity to archaeal PurA. However, how the transition of PurA to PurZ occurred during evolution is not clear; recapturing this process may shed light on the origin of Z-containing phages. Here we describe the computer-guided identification and biochemical characterization of a naturally existing PurZ variant, PurZ0, which uses guanosine triphosphate as the phosphate donor rather than the ATP used by PurZ. The atomic resolution structure of PurZ0 reveals a guanine nucleotide binding pocket highly analogous to that of archaeal PurA. Phylogenetic analyses suggest PurZ0 as an intermediate during the evolution of archaeal PurA to phage PurZ. Maintaining the balance of different purines necessitates further evolvement of guanosine triphosphate-using PurZ0 to ATP-using PurZ in adaptation to Z-genome life.
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Data availability
The coordinates and structure of GpPurZ0 in complex with GDP and phosphate have been deposited in the PDB under accession no. 7VF6. Other data (for example, bioinformatics) have been uploaded to a publicly available database (https://doi.org/10.6084/m9.figshare.22736654). Source data are provided with this paper.
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Acknowledgements
We thank the instrument analytical centre of the School of Pharmaceutical Science and Technology at Tianjin University; in particular, we thank Y. Gao for providing assistance with the liquid chromatography-MS analysis and the Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine. This work was supported by the National Natural Science Foundation of China (NSFC) Distinguished Young Scholar of China Program award no. 32125002 to Y. Zhang, award no. 32225003 to M.L., NSFC award no. 32122024 to S.Z. and award no. 31971178 to S.Z., as well as the Innovation Team Project of the Universities in Guangdong Province award no. 2020KCXTD023 to M.L.
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Y.T., Y. Zhou and L.J. conducted all the biochemical experiments. Y.T., X.W., Y.L. and H.C. performed the bioinformatics analysis. Y.T., Y.L., M.L., S.Z. and Y. Zhang designed the experiments and wrote the paper.
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A patent application related to this work has been filed: patent no. WO2022/152192, enzyme involved in the synthesis of phage diaminopurine and use thereof (Y. Zhang, Y. Zhou and Y.T.). The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 AG balancing in purine nucleotide synthesis.
a, Synthesis of AMP and GMP from IMP. b, PurZ and PurB are used in the extended G branch pathway to make dZMP. c, Proposed molecular evolution route from PurA to PurZ. The energy donors (ATP or GTP) are labeled in red to highlight their importance in regulating AG balance.
Extended Data Fig. 2 SDS-PAGE gel analyses of GpPurZ0.
a, 4–20% SDS gel with lane 1, molecular weight marker; and lanes 2, 3 and 4 with 1, 2, 4 μg of purified GpPurZ0. b, Elution profile of GpPurZ0 using Superdex 200 gel filtration chromatography. c, Calibration plot based on the molecular weight standards, including horse apoferritin (443 kDa), sweet potato β-Amylase (200 kDa), BSA (66 kDa), and bovine carbonic anhydrase (29 kDa) (Sigma MWGF 1000-1KT).
Extended Data Fig. 3 Spectrometric assays of GpPurZ0 to determine substrate specificity.
a-p, Different substrate combinations were used as substrates as indicated.
Extended Data Fig. 4 Enzyme kinetic assays of GpPurZ0.
Kinetic assays were performed by monitoring increase of absorbance at 287 nm with varying concentrations of one substrate as indicated on X-axis, a-d, GTP, dGTP, dGMP and dIMP respectively, while maintaining the other two substrates in excess. Data are mean ± SEM (N = 3 replicates).
Extended Data Fig. 5 dGMP/IMP binding site comparison.
a, dGMP binding site comparison between GpPurZ0 (pink, 7VF6) and CpPurZ (light green, 7ODX). b, dGMP binding site comparison between GpPurZ0 (pink, 7VF6) and VpPurZ (turquoise, 6FM1). c, dGMP/IMP binding site comparison between GpPurZ0 (pink, 7VF6) and PhPurA (blue, 5K7X).
Extended Data Fig. 6 Structure and sequence analyses of the NTP binding pocket in PurZ0, PurZ and PurA.
a, The NTP binding site of the EcPurA (PDB: 1CIB). b, Sequence logos of residues interacting with the purine moiety of NTPs in all PurA of bacteria. c, The NTP binding site of the GpPurZ0 (PDB: 7VF6). d, Sequence logos of residues interacting with the purine moiety of NTPs in PurZ0 of phages. e, The NTP binding site of the VpPurZ (PDB: 6FKO). f, Sequence logos of residues interacting with the purine moiety of NTPs in PurZ of phages.
Extended Data Fig. 7 Gel analyses and enzyme activity assays of different GpPurZ0 mutants.
a, Gel analysis of purified proteins. 4–20% SDS gel with lane 1, molecular weight marker; and lanes 2–7 with purified M304A, M304F, D306A, D306N, R20A, S15D mutants. b, Gel analysis of fractions during purification of the D306A mutant. 4–20% SDS gel with lane 1, molecular weight marker; lane 2, cell debris and inclusion body; lane 3, soluble fraction of total cell lysate; lane 4, unbound of the Ni-NTA column; lane 5, eluate with buffer containing 25 mM imidazole; lane 6, eluate with buffer containing 250 mM imidazole; lane 7, unbound of the DEAE column, and lane 8, eluate with buffer containing 400 mM KCl from the DEAE column. c, Enzyme activities of the mutants relative to that of the wild type enzyme. Data are mean ± SEM (N = 3 replicates). d-l, Spectrometric assays of the wild type and mutant GpPurZ0 enzymes.
Extended Data Fig. 8 Kinetic assays of the mutant enzymes.
a-c, D306N, M304F and R20A respectively. Data are mean ± SEM (N = 3 replicates).
Extended Data Fig. 9 Characterization of PurZ0 enzymes of different origins.
a-d, Gel analysis of the purified enzymes. 4–20% SDS gel with lane 1, molecular weight marker; and lanes 2, 3 and 4 with 1, 2, 4 μg of purified SpPurZ0, MptPurZ0, MpsPurZ0, MsPurZ0 respectively. e–h, Spectrometric assays of PurZ0 enzymes as indicated. i-l, PurZ0-catalyzed phosphate release detected by the phosphomolybdate assay; (+): the complete assay with GTP-dGMP, Asp and PurZ0 (-): without PurZ0, Asp (-): without Asp-Na+ as controls.
Extended Data Fig. 10
Genome neighborhood analyses of PurZ and PurZ0.
Supplementary information
Supplementary Information
Supplementary Figs. 1 and 2, Tables 1–4 and references.
Supplementary Data 1
Preliminary full worldwide PDB X-ray structure validation report.
Supplementary Data 2
Company names and catalogue numbers of the commercial reagents.
Source data
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Source Data Extended Data Fig. 2
Unprocessed gels and statistical source data.
Source Data Extended Data Fig. 3
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Source Data Extended Data Fig. 7
Unprocessed gels and statistical source data.
Source Data Extended Data Fig. 8
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Source Data Extended Data Fig. 9
Unprocessed gels and statistical source data.
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Tong, Y., Wu, X., Liu, Y. et al. Alternative Z-genome biosynthesis pathway shows evolutionary progression from Archaea to phage. Nat Microbiol 8, 1330–1338 (2023). https://doi.org/10.1038/s41564-023-01410-1
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DOI: https://doi.org/10.1038/s41564-023-01410-1