Molecular Mechanisms Modulating Glutamate Kinase Activity. Identification of the Proline Feedback Inhibitor Binding Site

Pérez-Arellano, Isabel; Carmona-Álvarez, Francisco; Gallego, José; Cervera, Javier

doi:10.1016/j.jmb.2010.10.019

Cited by 26 publications

(42 citation statements)

References 52 publications

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“…The crystal structure of the E. coli ␥-glutamyl kinase (49,51) revealed that the glutamate substrate binding site partially overlaps with that of the feedback inhibitor proline. The interactions of both molecules with the ␥-glutamyl kinases protein is modulated by a 16-amino-acid long flexible loop that links ␤-sheet 4 with ␣-helix E (49).…”

Section: Discussionmentioning

confidence: 99%

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Osmotically Controlled Synthesis of the Compatible Solute Proline Is Critical for Cellular Defense of Bacillus subtilis against High Osmolarity

et al. 2011

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Bacillus subtilis is known to accumulate large amounts of the compatible solute proline via de novo synthesis as a stress protectant when it faces high-salinity environments. We elucidated the genetic determinants required for the osmoadaptive proline production from the precursor glutamate. This proline biosynthesis route relies on the proJ-encoded ␥-glutamyl kinase, the proA-encoded ␥-glutamyl phosphate reductase, and the proH-encoded ⌬ 1 -pyrroline-5-caboxylate reductase. Disruption of the proHJ operon abolished osmoadaptive proline production and strongly impaired the ability of B. subtilis to cope with high-osmolarity growth conditions. Disruption of the proA gene also abolished osmoadaptive proline biosynthesis but caused, in contrast to the disruption of proHJ, proline auxotrophy. Northern blot analysis demonstrated that the transcription of the proHJ operon is osmotically inducible, whereas that of the proBA operon is not. Reporter gene fusion studies showed that proHJ expression is rapidly induced upon an osmotic upshift. Increased expression is maintained as long as the osmotic stimulus persists and is sensitively linked to the prevalent osmolarity of the growth medium. Primer extension analysis revealed the osmotically controlled proHJ promoter, a promoter that resembles typical SigA-type promoters of B. subtilis. Deletion analysis of the proHJ promoter region identified a 126-bp DNA segment carrying all sequences required in cis for osmoregulated transcription. Our data disclose the presence of ProA-interlinked anabolic and osmoadaptive proline biosynthetic routes in B. subtilis and demonstrate that the synthesis of the compatible solute proline is a central facet of the cellular defense to high-osmolarity surroundings for this soil bacterium.The soil-dwelling bacterium Bacillus subtilis (20) is frequently exposed to osmotic fluctuations in its environment as a consequence of wetting and drying cycles of the upper layers of the soil (8). As a result of these changes in the osmolarity and salinity of the habitat, the B. subtilis cell has to cope with osmotically instigated water fluxes across the cytoplasmic membrane. Consequently, the integrity of the cell is threatened under hypo-osmotic conditions, or its growth is impaired under hyperosmotic conditions (8). No microorganism can actively pump water in or out of the cell to compensate for water fluxes caused by changes in the external osmotic condition. Instead, microorganisms determine the direction and scale of water permeation across the cytoplasmic membrane indirectly by actively controlling the osmotic potential of their cytoplasm (9,66). This is often accomplished under high-osmolarity conditions by first importing large amounts of potassium as an emergency stress reaction (9,41,66). Subsequently, the cell replaces part of the accumulated potassium by a distinct class of highly water-soluble organic compounds, the compatible solutes (11). These osmolytes have been specifically selected in the course of evolution as effective osmo-and cytoprote...

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Section: Discussionmentioning

confidence: 99%

“…1D). The anabolic proline biosynthesis pathway in microorganisms is frequently regulated biochemically through allosteric feedback inhibition of the activity of the first proline-biosynthetic enzyme (ProB) by the end product proline (16,23,45,(49)(50)(51).…”

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confidence: 99%

Osmotically Controlled Synthesis of the Compatible Solute Proline Is Critical for Cellular Defense of Bacillus subtilis against High Osmolarity

et al. 2011

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“…The structure of bifunctional P5CS has not been reported, but individual structures of GK and GPR are available from bacteria (79,89). Important residues for glutamate binding in the GK domain are conserved among GK of different species (93,94). Proline biosynthesis is feedback inhibited by proline binding to the GK domain and interfering with the glutamate binding site (93).…”

Section: Proline Metabolic Enzymesmentioning

confidence: 99%

“…Important residues for glutamate binding in the GK domain are conserved among GK of different species (93,94). Proline biosynthesis is feedback inhibited by proline binding to the GK domain and interfering with the glutamate binding site (93).…”

Section: Proline Metabolic Enzymesmentioning

confidence: 99%

“…The feedback inhibition of P5CS is similar to that of bacterial GKs and involves competitive inhibition by proline with regard to glutamate (93,94). Structural analysis and site-directed mutagenesis of bacterial GKs suggest that proline partially occupies the glutamate binding site when bound to GK, thus interfering with glutamate binding and inhibiting GK activity (93). Incorporating a GK variant that is insensitive to proline inhibition has been used to overproduce proline in bacteria (23, 119) and yeast (125).…”

Section: Proline Biosynthesismentioning

confidence: 99%

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Proline Mechanisms of Stress Survival

Liang

Zhang

Natarajan

et al. 2013

Antioxidants & Redox Signaling

928

576

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Significance: The imino acid proline is utilized by different organisms to offset cellular imbalances caused by environmental stress. The wide use in nature of proline as a stress adaptor molecule indicates that proline has a fundamental biological role in stress response. Understanding the mechanisms by which proline enhances abiotic/biotic stress response will facilitate agricultural crop research and improve human health. Recent Advances: It is now recognized that proline metabolism propels cellular signaling processes that promote cellular apoptosis or survival. Studies have shown that proline metabolism influences signaling pathways by increasing reactive oxygen species (ROS) formation in the mitochondria via the electron transport chain. Enhanced ROS production due to proline metabolism has been implicated in the hypersensitive response in plants, lifespan extension in worms, and apoptosis, tumor suppression, and cell survival in animals. Critical Issues: The ability of proline to influence disparate cellular outcomes may be governed by ROS levels generated in the mitochondria. Defining the threshold at which proline metabolic enzyme expression switches from inducing survival pathways to cellular apoptosis would provide molecular insights into cellular redox regulation by proline. Are ROS the only mediators of proline metabolic signaling or are other factors involved? Future Directions: New evidence suggests that proline biosynthesis enzymes interact with redox proteins such as thioredoxin. An important future pursuit will be to identify other interacting partners of proline metabolic enzymes to uncover novel regulatory and signaling networks of cellular stress response. Antioxid. Redox Signal. 19, 998-1011.

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Effects of a novel variant of the yeast γ-glutamyl kinase Pro1 on its enzymatic activity and sake brewing

Murakami

Kotaka

Isogai

et al. 2020

Journal of Industrial Microbiology and Biotechnology

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Sake is a traditional Japanese alcoholic beverage brewed with the yeast Saccharomyces cerevisiae. Sake taste is affected by sugars, organic acids, and amino acids. We previously isolated mutants resistant to the proline analogue azetidine-2-carboxylate derived from a diploid sake yeast strain. Some of the mutants produced a greater amount of proline in the brewed sake. One of them (strain K-9-AZC) carried a novel mutation in the PRO1 gene encoding the Gln79His variant of the γ-glutamyl kinase Pro1, a key enzyme in proline biosynthesis in S. cerevisiae. This mutation resulted in extreme desensitization to feedback inhibition by proline, leading to proline overproduction. Interestingly, sake brewed with K-9-AZC contained 3.7-fold more proline, but only 25% less succinate than sake brewed with the parent strain. Metabolome analysis suggests that the decrease in succinate was attributable to a lower level of 2-oxoglutarate, which is converted into glutamate. The approach here could be a practical method for breeding of yeast strains involved in the diversity of sake taste.

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Molecular Mechanisms Modulating Glutamate Kinase Activity. Identification of the Proline Feedback Inhibitor Binding Site

Cited by 26 publications

References 52 publications

Osmotically Controlled Synthesis of the Compatible Solute Proline Is Critical for Cellular Defense of Bacillus subtilis against High Osmolarity

Osmotically Controlled Synthesis of the Compatible Solute Proline Is Critical for Cellular Defense of Bacillus subtilis against High Osmolarity

Proline Mechanisms of Stress Survival

Effects of a novel variant of the yeast γ-glutamyl kinase Pro1 on its enzymatic activity and sake brewing

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