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A counter-enzyme complex regulates glutamate metabolism in Bacillus subtilis

Nature Chemical Biology volume 18, pages 161–170 (2022)Cite this article

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Abstract

Multi-enzyme assemblies composed of metabolic enzymes catalyzing sequential reactions are being increasingly studied. Here, we report the discovery of a 1.6 megadalton multi-enzyme complex from Bacillus subtilis composed of two enzymes catalyzing opposite (‘counter-enzymes’) rather than sequential reactions: glutamate synthase (GltAB) and glutamate dehydrogenase (GudB), which make and break glutamate, respectively. In vivo and in vitro studies show that the primary role of complex formation is to inhibit the activity of GudB. Using cryo-electron microscopy, we elucidated the structure of the complex and the molecular basis of inhibition of GudB by GltAB. The complex exhibits unusual oscillatory progress curves and is necessary for both planktonic growth, in glutamate-limiting conditions, and for biofilm growth, in glutamate-rich media. The regulation of a key metabolic enzyme by complexing with its counter enzyme may thus enable cell growth under fluctuating glutamate concentrations.

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Fig. 1: GudB interacts with GltAB in glutamate-poor growth conditions.
Fig. 2: The phenotypic effects of GltAB and GudB and their interaction.
Fig. 3: Enzymatic kinetics of the GltAB–GudB complex.
Fig. 4: The stoichiometry, oligomeric state and atomic structure of the GltAB–GudB complex.
Fig. 5: The structural basis for inhibition of GudB by GltA binding.
Fig. 6: GudB–GltAB interaction is important for biofilm formation.

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Data availability

The proteomics data are provided as Excel sheets (Supplementary Datasets 1 and 2). The structural data are deposited in the PDB (PDB codes 7MFM and 7MFT). Source data are provided with this paper.

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Acknowledgements

We thank K.P. Cherukuri for help in synthesis of DSG, A. Leytens for assistance in cloning and Y. Kushmaro for screening different crosslinkers. We are grateful to M. Kupervaser and Y. Levin from The De Botton Protein Profiling institute of the Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, for the proteomics analysis. We thank A. Sonn Segev and M. Goldsmith for their assistance with the mass photometry experiments. We thank S. Malitsky and M. Itkin for the metabolomics analysis. We thank U. Sauer, U. Alon, N. Tokuriki and S. Laxman for their critical feedback on the paper. We thank F. Jonas for valuable comments on the paper and B. Ross for proofreading. Funding by the Israel Science Foundation grant no. 2575/20 is gratefully acknowledged. M.S. is the incumbent of the Aharon and Ephraim Katzir Memorial Professorial Chair. The research by S.V. is supported by the Clore Israel Foundation. J.S.F. is supported by National Institutes of Health (NIH) grant no. GM123159 and D.J.L. is supported by NIH grant no. F32 AI148120. D.S.T. is the incumbent of the Nella and Leon Benoziyo Professorial Chair.

Author information

Author notes

  1. These authors contributed equally: D. John Lee, Nadav Elad.

  2. Deceased: Dan S. Tawfik.

Authors and Affiliations

  1. Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel

    Vijay Jayaraman, Shay Vimer, Michal Sharon & Dan S. Tawfik

  2. Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA, USA

    D. John Lee & James S. Fraser

  3. Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel

    Nadav Elad

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Contributions

D.S.T. conceptualized the study and supervised all research. V.J. performed all experimental work unless otherwise stated. S.V. performed the native MS and M.S. supervised this analysis. N.E. acquired the cryo-EM data. D.J.L., V.J. and N.E. performed the cryo-EM data processing and model building. This was supervised by J.S.F. V.J., J.S.F. and D.S.T wrote the paper and all authors reviewed the data and the paper.

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Correspondence to Vijay Jayaraman or James S. Fraser.

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Extended data

Extended Data Fig. 1 GudB interacts with GltAB.

(a) Western blot analysis using an anti-GudB antibody on lysates prepared from DSG treated B. subtilis cells grown on different C/N source. The high molecular weight species of GudB are seen only in cells grown in glucose−ammonia. (b) Western blot analysis using a Strep-Tactin-HRP antibody on the same samples used in A. Here the antibody was used on lysates prepared from both DSG-treated (lanes right to the molecular weight marker) and untreated cells (lanes left to the marker). As, can been seen, GltB is expressed only in cells grown on glucose−ammonia, and under this condition, it is also part of a high-MW complex (in the DSG treated sample) (Fig. 1b). (c) Enzyme kinetics of wild-type GltAB (orange) and its mutant GltABC1A (light orange), show that the mutant is completely inactive. A minor decrease in absorbance with mutant GltA is due to non-enzymatic oxidation of NADPH. The reaction mixture consisted of 2 mM AKG, 5 mM glutamine, 200 µM NADPH, 5 mM DTT, 5 mM MgSO4. The reaction was initiated with 2.5 µg of the wild-type or mutant enzyme. (d) SDS-PAGE of the eluate from Strep-Tactin column to which lysate from B. subtilis strain expressing GltABC1A in the background of constitutively expressed GudB or RocG is applied. Co-elution of GudB along with the inactive GltA (GltABC1A) upon pulldown of Strep-GltB (PgudB-gudB lane), while GltABC1A elutes on its own when expressed with RocG (PgudB-rocG lane). Images in a-d are obtained from a single experiment and are a representative of at least 2 independent experiments.

Source data

Extended Data Fig. 2 Phenotyping of Phs-gltAB strain.

The strain expresses GltAB from the IPTG- inducible hyperspank (Phs) promoter. Without IPTG, the strain cannot grow in minimal medium containing glucose−ammonia as the C/N source (light orange); however, addition of 500 µM IPTG restores growth to almost wild-type levels (orange). Addition of IPTG does not have any effect on the growth of the parental wild-type strain. n = 3 are three independent measurements. Data is presented as mean of all measurements and error bars represent SD.

Source data

Extended Data Fig. 3 Steady-state kinetics of GltAB, and GudB–GltAB complex.

(a-f) Substrate titration plots for the glutamate synthase activity of GltAB by itself (A-C) and in the GudB bound form (D-F). One of the substrates was titrated while keeping the rest saturating: glutamine (5 mM), AKG (α-ketoglutarate, 2 mM), NADPH (200 µM). n = 2 are two independent experiments. Data is presented as mean and error bars indicate SD. (g) AKG and NADPH promote the assembly of GltA and GltB. Only 5 % of the counts corresponded to GltAB when no ligands were added (left panel). Addition of AKG increase the GltAB count to 12 % (Middle panel) and the presence of NADPH in addition to AKG increased the GltAB counts to 16 % (right panel). (h-l) Non-hyperbolic progress curves displayed by GudB-GltAB complex in the presence of all the substrates except AKG. While at high glutamate concentrations GudB’s activity dominates (K), at low glutamate concentration the synthase activity takes over (G). At intermediate concentration of glutamate (H- J) the progress curve oscillates between GudB- and GltAB-predominant phases. (m) Progress curves obtained using different amount of the GudB-GltAB complex at 37.5 mM glutamate (where oscillations are most pronounced; panel i). The reaction mixture contained all substrates for GudB and GltAB except AKG. (n) Plotted are the slopes derived from phase 2 of the progress curves (GudB’s activity as shown in the inset) as a function of the complex concentration. The non-linear relationship suggest that association-dissociation of the complex plays a role in turning off-on GudB’s activity. Note also the elapsed time for phase 2 (the 2nd GudB activity phase) that becomes longer as the complex concentration decreases. The inset shows the phase 3 in each of the individual progress curves. (o) Unlike phase 2, phase 3 that corresponds to GltAB’s activity shows linear dependence with enzyme concentration. The inset shows phase 3 in each of the individual progress curves. Data in panels g-o are from single experiment and are representative of at least 2 independent experiments.

Source data

Extended Data Fig. 4 Cyro-EM image processing for GudB6-GltA6B6.

(a) Scheme for single particle cryo-EM analysis of the GudB6-GltA6B6. Details of the process are described in the Methods section. Briefly, particles were iteratively picked from selected micrographs using well resolved 2D class averages, followed by Ab initio 3D reconstruction and classification into five classes. The best resolved 3D class was refined with D3 symmetry imposed. In order to account for deviations from D3 symmetry, further refinement focused on single GudB-GltAB asymmetric units. The number of particles that are included in the maps are indicated, along with the estimated resolution where relevant. (b) FSC curves for the refined GudB-GltAB asymmetric map. (c) Angular distribution plot. (d) 3D map colored according to local resolution estimate.

Extended Data Fig. 5 Native-MS and the corresponding particle types observed in cryo-EM of the GudB enriched preparation of the GudB-GltAB complex.

(a) Native-MS spectra showing different species of GudB-GltAB complex. These species primarily differ in the number of GltAB heterodimers attached to the GudB hexamer (from 3-6). Charge states (z) and the difference from theoretical mass (ΔT) is indicated for each species. While the mass of the fully assembled complex agreed well with the expected mass, high ΔT values of the other species could be because of degradation of some of the component proteins during the extended incubation step with E. coli lysate (Methods section) during the purification process. The inset shows the SDS-PAGE of GudB-GltAB complex used for the native-MS. The sample was prepared after enriching the B. subtilis lysate with recombinant GudB expressed and purified from E. coli (see Protein expression and purification, Methods). The image is from a single experiment and is a representative of at least two independent experiments. This sample contained a higher fraction of GudB and was also used for cryo-EM to obtain the high-resolution structure of GudB6-GltA2B2 complex (Fig. 5). (b) Preliminary cryo-EM maps corresponding to different particle types (GudB6-GltA2-4B2-4) observed. The key difference between particles is the number of GltAB subunits - the least being two (grey) and the maximum four (violet).

Source data

Extended Data Fig. 6 Single particle cryo-EM analysis of the GudB6-GltA2B2.

(a) The particles were iteratively picked from selected micrographs and classified. All non-ice particles were carried forward and subjected to a two-class 3D auto refinement in cisTEM using the preliminary GudB6-GltA2B2 reference. This yielded one noise class carrying unaligned particles and high frequency noise, and one class representing clear density for the GudB6-GltA2B2 species. This class was refined using auto and manual methods in cisTEM, with C2 symmetry applied. The number of particles and the estimated resolution are indicated. (b) FSC curves for the refined GudB6-GltA2B2 map. (c) Angular distribution plot and local resolution estimation (Relion3.1.2).

Extended Data Fig. 7 Effect of GltA binding on GudB.

(a) The co-factors in GltAB: FAD, two 4Fe-4S clusters (SF4), 3Fe-4S cluster (F3S) and FMN. These co-factors are involved in shuttling of electrons from NADPH to 2-iminoglutarate along shown arrow. GltA and GltB are shown in a transparent surface representation. (b) GltAB binding captures GudB in an ‘open’ state with the distance of 29.1 Å between residues R280 (in the co-factor binding domain, shown in light green) and K122 (in the substrate binding domain, shown in green). These residues are equivalent to R271 and R124 in the model of a ‘super-closed’ glutamate dehydrogenase (PDB 5XVX)30 (right panel). (c) GltAB in sub-stoichiometric amounts promotes hexamerization of GudB by interacting with multiple GudB protomers (from different dimers, as shown in Fig. 5c) and hence prevents loss of activity. The assay buffer contained 400 mM glutamate and 4 mM NAD+ and the reaction was initiated by the addition of an enzyme mix consisting of GudB pre-incubated with different amounts of GltAB (in all the reactions, GudB was present at a final concentration is 0.05 µM). GltAB prevents GudB inactivation in a concentration dependent manner. Individual data points are shown from two independent measurements and error bars indicate standard deviation of the mean.

Source data

Extended Data Fig. 8 Details of the GudB-GltAB interaction.

(a) Key interactions between GltA (ovals on orange line) and two GudB protomers (ovals on green/major interaction and yellow line/minor interaction); salt bridges are shown in red and hydrogen bonds in blue. The border of ovals are colored based on domain/structural feature to which the residue belongs. (b) Binding to GltA stabilizes many loops in the cofactor binding domain of GudB. Shown are a GltA-bound GudB protomer (left, chain A in 7MFM) and a free GudB protomer in the same structure (right, chain B) in the ‘putty’ representation as implemented in PyMol. The radius of the ribbon increases from low to high B-factor and the Cα B-factors are shown in dark blue (lowest B-factor, 54) to red (highest B-factor, 163). nding site is located about 45 Å and 75 Å from the regulatory loop of GltA.

Extended Data Fig. 9 Biofilm growth and disruption phenotypes.

(a) Biofilm diameters measured at different time points. While ΔgudB biofilms were bigger in size and grew faster than wild-type biofilms, ΔgltA and ΔgltB biofilms were smaller. The dashed lines at 2.5 mm and 35 mm indicates the starting size of the biofilm, and the diameter of the well, used to grow the biofilm, respectively. The measurements were from four independent experiments (n = 4). Data is shown as mean and error bars represent standard deviation. (b) Similarity in biofilm morphology between ΔgudB and the Phs-gltAB strains (grown with 100 µM IPTG). Overexpression of GltAB in the latter increases in synthase activity and also silences GudB, thereby resembling the ΔgudB biofilm morphology. Both biofilms grew rapidly and had large wrinkles spreading from the interior to the periphery of the biofilm. All the images in this panel are reproduced from Fig. 6 for better representation of the biofilm morphology. (c) Similarity in biofilm morphology between ΔgltB and the Phs-gltABC1A strains (grown with 100 µM IPTG). In both the biofilms the wrinkles are restricted to the interior of the biofilm.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–7.

Reporting Summary

Supplementary Data 1

Proteomics analysis of the GudB pulldown with and without the presence of crosslinker.

Source data

Source Data Fig. 1

Unprocessed gels and western blot.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 3

Unprocessed gels.

Source Data Fig. 4

Unprocessed gels.

Source Data Extended Data Fig. 1

Unprocessed western blot and silver stained gel.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 5

Unprocessed gel.

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Statistical source data.

Source Data Extended Data Fig. 9

Statistical source data.

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Jayaraman, V., Lee, D.J., Elad, N. et al. A counter-enzyme complex regulates glutamate metabolism in Bacillus subtilis. Nat Chem Biol 18, 161–170 (2022). https://doi.org/10.1038/s41589-021-00919-y

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  • DOI: https://doi.org/10.1038/s41589-021-00919-y

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