Structural basis for the hyperthermostability of an archaeal enzyme induced by succinimide formation

Dongre, A.; Das, Sudip Kumar; Bellur, Asutosh; Kumar, Sanjeev; Chandrashekarmath, Anusha; Karmakar, Tarak; Balaram, Padmanabhan; Balasubramanian, Sundaram; Balaram, Hemalatha

doi:10.1016/j.bpj.2021.07.014

Cited by 5 publications

(7 citation statements)

References 102 publications

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“…We had earlier solved the structure of the MjGATase subunit, , and here, we report the crystal structure of the XMP complexed MjATPPase subunit solved to a resolution of 2.1 Å (Table ). The model has four chains in the asymmetric unit with chains A and B, and C and D forming two homodimers, and each protomer bound to a molecule of XMP (Figure A and Figure S1A).…”

Section: Resultsmentioning

confidence: 95%

“…(A) Inter-domain interface in two-domain type GMP synthetases with GATase in the 0° rotated state as exemplified by the PfGMPS/XMP structure. The MjGATase structure (PDB ID: 7D40 ) is superposed on the GATase domain of PfGMPS/XMP. The N-terminal residues of MjGATase, namely, 1, 20, and 27, which are involved in 14 inter-subunit cross-links, are shown as spheres.…”

Section: Discussionmentioning

confidence: 99%

“…We observed 16 unique MjGATase intralinks in the heterodimer as well as the heterotetramer with 12 common to the two sets (Figure 6A and Table S3 and Supporting Information 2. Structural validation of these intralinks was performed by measuring the Euclidean distances between the Cα atoms of the cross-linked residues in the apo structure of MjGATase (PDB ID: 7D40 61 ). The distances of the 16 GATase intralinks were within a cut-off limit of 30 Å imposed by the length of the crosslinker arm (Figure 6B and Table S3).…”

Section: Mjatppase Displays Unusually High Affinity For Nh 4 CL and H...mentioning

confidence: 99%

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Mechanistic Insights into the Functioning of a Two-Subunit GMP Synthetase, an Allosterically Regulated, Ammonia Channeling Enzyme

et al. 2022

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Guanosine 5′-monophosphate (GMP) synthetases, enzymes that catalyze the conversion of xanthosine 5′-monophosphate (XMP) to GMP, are composed of two different catalytic units, which are either two domains of a polypeptide chain or two subunits that associate to form a complex. The glutamine amidotransferase (GATase) unit hydrolyzes glutamine generating ammonia, and the ATP pyrophosphatase (ATPPase) unit catalyzes the formation of an AMP-XMP intermediate. The substrate-bound ATPPase allosterically activates GATase, and the ammonia thus generated is tunneled to the ATPPase active site where it reacts with AMP-XMP generating GMP. In ammonia channeling enzymes reported thus far, a tight complex of the two subunits is observed, while the interaction of the two subunits of Methanocaldococcus jannaschii GMP synthetase (MjGMPS) is transient with the underlying mechanism of allostery and substrate channeling largely unclear. Here, we present a mechanistic model encompassing the various steps in the catalytic cycle of MjGMPS based on biochemical experiments, crystal structure, and cross-linking mass spectrometry guided integrative modeling. pH dependence of enzyme kinetics establishes that ammonia is tunneled across the subunits with the lifetime of the complex being ≤0.5 s. The crystal structure of the XMP-bound ATPPase subunit reported herein highlights the role of conformationally dynamic loops in enabling catalysis. The structure of MjGMPS derived using restraints obtained from cross-linking mass spectrometry has enabled the visualization of subunit interactions that enable allostery under catalytic conditions. We integrate the results and propose a functional mechanism for MjGMPS detailing the various steps involved in catalysis.

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Section: Resultsmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Section: Mjatppase Displays Unusually High Affinity For Nh 4 CL and H...mentioning

confidence: 99%

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Mechanistic Insights into the Functioning of a Two-Subunit GMP Synthetase, an Allosterically Regulated, Ammonia Channeling Enzyme

et al. 2022

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“…We had earlier solved the structure of the MjGATase subunit by NMR 20 and subsequently by X-ray crystallography. 27 Here we report the crystal structure of XMP complexed MjATPPase subunit solved to a resolution of 2.1 Å (Table 1). The model has four chains in the asymmetric unit with chains A and B, and C and D forming two homodimers, and each protomer bound to a molecule of XMP (Figure 3A and S1A).…”

Section: Crystal Structure Of Mjatppasementioning

confidence: 99%

Mechanistic insights into the functioning of GMP synthetase: a two-subunit, allosterically regulated, ammonia tunnelling enzyme

Shivakumaraswamy

Kumar

Bellur

et al. 2022

Preprint

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Guanosine 5'-monophosphate (GMP) synthetases, enzymes that catalyze the conversion of xanthosine 5'-monophosphate (XMP) to GMP are comprised of two different catalytic units, which are either two domains of a polypeptide chain or two subunits that associate to form a complex. The glutamine amidotransferase (GATase) unit hydrolyzes glutamine generating ammonia and the ATP pyrophosphatase (ATPPase) unit catalyzes the formation of AMP-XMP intermediate. The substrate-bound ATPPase allosterically activates GATase and the ammonia thus generated is tunnelled to the ATPPase active site where it reacts with AMP-XMP generating GMP. In ammonia tunnelling enzymes reported thus far, a tight complex of the two subunits is observed, while the interaction of the two subunits of Methanocaldococcus jannaschii GMP synthetase (MjGMPS) is transient with the underlying mechanism of allostery and substrate channelling largely unclear. Here, we present a mechanistic model encompassing the various steps in the catalytic cycle of MjGMPS based on biochemical experiments, crystal structure and cross-linking mass spectrometry guided integrative modelling. pH dependence of enzyme kinetics establish that ammonia is tunnelled across the subunits with the lifetime of the complex being ≤ 0.5 s. The crystal structure of XMP-bound ATPPase subunit reported herein highlights the role of conformationally dynamic loops in enabling catalysis. The structure of MjGMPS derived using restraints obtained from cross-linking mass spectrometry has enabled the visualization of subunit interactions that enable allostery under catalytic conditions. We integrate the results and propose a functional mechanism for MjGMPS detailing the various steps involved in catalysis.

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“…In particular metadynamics has been widely applied for modeling (bio)chemical processes, such as the nucleation and growth of carbon nano-tubes, [39][40][41] proton transfers, [42][43][44] conformations of biomolecules in solution, 28,[45][46][47] as well as their binding at inorganic surfaces. [48][49][50] Also the herein utilized VES procedure [17][18][19][20][21][22][23] has been employed for modeling of molecular conformations in solutions, 18,51 crystal nucleation, 21,30 whereas the ability of VES to handle large sets of CVs have enabled simulations of protein folding.…”

Section: Introductionmentioning

confidence: 99%

Improved reweighting protocols for variationally enhanced sampling simulations with multiple walkers

Stevensson¹,

Edén²

2023

Phys. Chem. Chem. Phys.

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Structural basis for the hyperthermostability of an archaeal enzyme induced by succinimide formation

Cited by 5 publications

References 102 publications

Mechanistic Insights into the Functioning of a Two-Subunit GMP Synthetase, an Allosterically Regulated, Ammonia Channeling Enzyme

Mechanistic Insights into the Functioning of a Two-Subunit GMP Synthetase, an Allosterically Regulated, Ammonia Channeling Enzyme

Mechanistic insights into the functioning of GMP synthetase: a two-subunit, allosterically regulated, ammonia tunnelling enzyme

Improved reweighting protocols for variationally enhanced sampling simulations with multiple walkers

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