Enhanced and feedback-resistant γ-glutamyl kinase activity of an Escherichia coli transformant carrying a mutated proB gene of Streptococcus thermophilus

Massarelli, I

doi:10.1016/s0378-1097(99)00573-x

Cited by 9 publications

(21 citation statements)

References 21 publications

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“…3A). Interestingly the base changes, each of which results in an amino acid substitution within a defined (26-amino-acid) region of the GK enzyme, map closely to previously isolated mutations leading to proline overproduction in other genera (13,22,28,29,32,39) (Fig. 3B).…”

Section: Resultssupporting

confidence: 72%

“…1) indicated that as with the majority of systems (both prokaryotic and eukaryotic), listerial proline biosynthesis from glutamate may be regulated by proline-dependent feedback inhibition of the GK activity. Mutations leading to proline analogue resistance (and consequential proline hyperproduction) have been described for a number of organisms and have in each case been linked to mutations in GK, leading to a decreased sensitivity of the enzyme for its allosteric effector proline and its analogues (13,22,28,29,32).…”

Section: Resultsmentioning

confidence: 99%

“…Studies on purified enzymes suggest that in addition to proline-mediated inhibition, the ␥-glutamyl kinase activities of GK and P5CS are also modulated to a lesser extent by glutamate and ADP, thereby tuning proline synthesis to cellular substrate and energy availability (37, 39). Proline hyperproducing strains of bacteria, exhibiting reduced proline-mediated feedback inhibition of GK activity (a result of single-base-pair substitutions in either the bacterial proB gene [13,22,28,29,32] In addition to the obvious advantages for commercial amino acid synthesis (29), the osmoprotective properties of proline overproduction (19) have led to the development of transgenic drought-resistant plants (17). However, since proline may function as a potential virulence factor (2, 12, 33) and is known to facilitate the growth of certain pathogenic bacteria at elevated osmolarities (9), the use of transmissible genetic elements encoding proline hyperproduction may lead to undesirable consequences, if introduced prematurely into the natural environment.…”

mentioning

confidence: 99%

“…Studies on purified enzymes suggest that in addition to proline-mediated inhibition, the ␥-glutamyl kinase activities of GK and P5CS are also modulated to a lesser extent by glutamate and ADP, thereby tuning proline synthesis to cellular substrate and energy availability (37,39). Proline hyperproducing strains of bacteria, exhibiting reduced proline-mediated feedback inhibition of GK activity (a result of single-base-pair substitutions in either the bacterial proB gene [13,22,28,29,32] or the 5Ј domain of the plant P5CS coding region [39]), have been isolated based on their resistance to toxic proline analogues (L-azetidine-2-carboxylic acid [AZ] [15] and 3,4-dehydro-DL-proline, compounds which inhibit GK activity while not interfering with protein synthesis [23]). …”

mentioning

confidence: 99%

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Mutations in the Listerial proB Gene Leading to Proline Overproduction: Effects on Salt Tolerance and Murine Infection

Sleator

Gahan

Hill

2001

Appl Environ Microbiol

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The observed sensitivity of Listeria monocytogenes to the toxic proline analogue L-azetidine-2-carboxylic acid (AZ) suggested that proline synthesis in Listeria may be regulated by feedback inhibition of ␥-glutamyl kinase (GK), the first enzyme of the proline biosynthesis pathway, encoded by the proB gene. Taking advantage of the Epicurian coli mutator strain XL1-Red, we performed random mutagenesis of the recently described proBA operon and generated three independent mutations in the listerial proB homologue, leading to proline overproduction and salt tolerance when expressed in an E. coli (⌬proBA) background. While each of the mutations (located within a conserved 26-amino-acid region of GK) was shown to confer AZ resistance (AZ r ) on an L. monocytogenes proBA mutant, listerial transformants failed to exhibit the salt-tolerant phenotype observed in E. coli. Since proline accumulation has previously been linked to the virulence potential of a number of pathogenic bacteria, we analyzed the effect of proline overproduction on Listeria pathogenesis. However, our results suggest that as previously described for proline auxotrophy, proline hyperproduction has no apparent impact on the virulence potential of Listeria.Genetic and physiological analysis of proline accumulation in both prokaryotic and eukaryotic systems (11,20) has provided evidence that is consistent with diverse functions of proline, not only as a source of energy, carbon and nitrogen but also as an effective osmolyte (1, 10, 11, 23) and more recently as a potential virulence factor for a number of pathogenic bacteria (2,12,33).While proline can be synthesized from ornithine in both plants and animals (18), glutamate is the primary precursor for proline biosynthesis in bacteria (23) and in osmotically stressed plant cells (14). Bacterial proline synthesis from glutamate occurs via three enzymatic reactions, catalyzed by ␥-glutamyl kinase (GK) (proB product, EC 2.7.2.11), ␥-glutamyl phosphate reductase (GPR) (proA product, EC 1.2.1.41), and ⌬ 1 -pyrroline-5-carboxylate reductase (P5C) (proC product, EC 1.5.1.2). For the majority of bacteria the proB and proA genes constitute an operon, which is distant from proC on the chromosome. In plants, e.g., Vigna aconitifolia and Arabidopsis, the first two steps of proline biosynthesis from glutamate are catalyzed by ⌬ 1 -pyrroline-5-carboxylate synthetase (P5CS), a bifunctional enzyme with both GK and GPR activities at the Nand C-terminal domains, respectively (18).For both prokaryotic and eukaryotic systems, proline synthesis from glutamate is regulated by feedback inhibition of the first enzyme in the pathway. Studies on purified enzymes suggest that in addition to proline-mediated inhibition, the ␥-glutamyl kinase activities of GK and P5CS are also modulated to a lesser extent by glutamate and ADP, thereby tuning proline synthesis to cellular substrate and energy availability (37, 39). Proline hyperproducing strains of bacteria, exhibiting reduced proline-mediated feedback inhibition of GK activity (a resu...

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Section: Resultssupporting

confidence: 72%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

“…Studies on purified enzymes suggest that in addition to proline-mediated inhibition, the ␥-glutamyl kinase activities of GK and P5CS are also modulated to a lesser extent by glutamate and ADP, thereby tuning proline synthesis to cellular substrate and energy availability (37,39). Proline hyperproducing strains of bacteria, exhibiting reduced proline-mediated feedback inhibition of GK activity (a result of single-base-pair substitutions in either the bacterial proB gene [13,22,28,29,32] or the 5Ј domain of the plant P5CS coding region [39]), have been isolated based on their resistance to toxic proline analogues (L-azetidine-2-carboxylic acid [AZ] [15] and 3,4-dehydro-DL-proline, compounds which inhibit GK activity while not interfering with protein synthesis [23]). …”

mentioning

confidence: 99%

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Mutations in the Listerial proB Gene Leading to Proline Overproduction: Effects on Salt Tolerance and Murine Infection

Sleator

Gahan

Hill

2001

Appl Environ Microbiol

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“…In a site-directed mutagenesis of the V. aconitifolia P5CS, Zhang et al (22) showed that amino acid substitutions at positions 126 and 129 in the V. aconitifolia P5CS abolished proline feedback regulation. Other single amino acid mutations involving feedback regulation of GK have been recently reported in Streptococcus thermophilus and Listeria monocytogenes (26,27). In all, eight amino acid residues have been identified in six organisms as important for proline feedback regulation of GK (22-24, 26 -28, 32).…”

mentioning

confidence: 90%

Identification of Regions of the Tomato γ-Glutamyl Kinase That Are Involved in Allosteric Regulation by Proline

Fujita¹,

Maggio²,

Garcia-Rios³

et al. 2003

Journal of Biological Chemistry

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The first step of proline biosynthesis is catalyzed by ␥-glutamyl kinase (GK). To better understand the feedback inhibition properties of GK, we randomly mutagenized a plasmid carrying tomato tomPRO1 cDNA, which encodes proline-sensitive GK. A pool of mutagenized plasmids was transformed into an Escherichia coli GK mutant, and proline-overproducing derivatives were selected on minimal medium containing the toxic proline analog 3,4-dehydro-DL-proline. Thirty-two mutations that conferred 3,4-dehydro-DL-proline resistance were obtained. Thirteen different single amino acid substitutions were identified at nine different residues. The residues were distributed throughout the Nterminal two-thirds of the polypeptide, but 9 mutations affecting 6 residues were in a cluster of 16 residues. GK assays revealed that these amino acid substitutions caused varying degrees of diminished sensitivity to proline feedback inhibition and also resulted in a range of increased proline accumulation in vivo. GK belongs to a family of amino acid kinases, and a predicted threedimensional model of this enzyme was constructed on the basis of the crystal structures of three related kinases. In the model, residues that were identified as important for allosteric control were located close to each other, suggesting that they may contribute to the structure of a proline binding site. The putative allosteric binding site partially overlaps the dimerization and substrate binding domains, suggesting that the allosteric regulation of GK may involve a direct structural interaction between the proline binding site and the dimerization and catalytic domains.

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Quinoline Derivatives Kill Mycobacterium tuberculosis by Activating Glutamate Kinase

Makafe

Hussain

Surineni

et al. 2019

Cell Chemical Biology

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Enhanced and feedback-resistant γ-glutamyl kinase activity of an Escherichia coli transformant carrying a mutated proB gene of Streptococcus thermophilus

Cited by 9 publications

References 21 publications

Mutations in the Listerial proB Gene Leading to Proline Overproduction: Effects on Salt Tolerance and Murine Infection

Mutations in the Listerial proB Gene Leading to Proline Overproduction: Effects on Salt Tolerance and Murine Infection

Identification of Regions of the Tomato γ-Glutamyl Kinase That Are Involved in Allosteric Regulation by Proline

Quinoline Derivatives Kill Mycobacterium tuberculosis by Activating Glutamate Kinase

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