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Marie-jöelle Virolle - One of the best experts on this subject based on the ideXlab platform.
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Expression of genes of the Pho regulon is altered in Streptomyces Coelicolor.
Scientific reports, 2020Co-Authors: Aaron Millan-oropeza, Celine Henry, Clara Lejeune, Michelle David, Marie-jöelle VirolleAbstract:Most currently used antibiotics originate from Streptomycetes and phosphate limitation is an important trigger of their biosynthesis. Understanding the molecular processes underpinning such regulation is of crucial importance to exploit the great metabolic diversity of these bacteria and get a better understanding of the role of these molecules in the physiology of the producing bacteria. To contribute to this field, a comparative proteomic analysis of two closely related model strains, Streptomyces lividans and Streptomyces Coelicolor was carried out. These strains possess identical biosynthetic pathways directing the synthesis of three well-characterized antibiotics (CDA, RED and ACT) but only S. Coelicolor expresses them at a high level. Previous studies established that the antibiotic producer, S. Coelicolor, is characterized by an oxidative metabolism and a reduced triacylglycerol content compared to the none producer, S. lividans, characterized by a glycolytic metabolism. Our proteomic data support these findings and reveal that these drastically different metabolic features could, at least in part, due to the weaker abundance of proteins of the two component system PhoR/PhoP in S. Coelicolor compared to S. lividans. In condition of phosphate limitation, PhoR/PhoP is known to control positively and negatively, respectively, phosphate and nitrogen assimilation and our study revealed that it might also control the expression of some genes of central carbon metabolism. The tuning down of the regulatory role of PhoR/PhoP in S. Coelicolor is thus expected to be correlated with low and high phosphate and nitrogen availability, respectively and with changes in central carbon metabolic features. These changes are likely to be responsible for the observed differences between S. Coelicolor and S. lividans concerning energetic metabolism, triacylglycerol biosynthesis and antibiotic production. Furthermore, a novel view of the contribution of the bio-active molecules produced in this context, to the regulation of the energetic metabolism of the producing bacteria, is proposed and discussed.
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Quantitative proteomics analysis confirmed oxidative metabolism predominates in Streptomyces Coelicolor versus glycolytic metabolism in Streptomyces lividans
Journal of Proteome Research, 2017Co-Authors: Aaron Millan-oropeza, Celine Henry, Melisande Blein-nicolas, Anne Aubert, Fathi Moussa, Jean Bleton, Marie-jöelle VirolleAbstract:Recent physiological studies indicated that S. lividans metabolism was mainly glycolytic, whereas S. Coelicolor Metabolism was mainly oxidative. To determine whether such metabolic characteristics were correlated with consistent proteomics features, a comparative label-free, shotgun proteomics analysis of these strains was carried out. Among 2024 proteins identified; 360 showed significant differences in abundance between the strains. This study revealed that S. Coelicolor catabolized glucose less actively than S. lividans, whereas the amino acids present in the medium were catabolized less actively by S. lividans than by S. Coelicolor. The abundance of glycolytic proteins in S. lividans was consistent with its high glycolytic activity, whereas the abundance of proteins involved in the catabolism of amino acids in S. Coelicolor provided an explanatory basis for its predominantly oxidative metabolism. In this study, conducted under conditions of low O-2 availability, proteins involved in resistance to oxidative stress and those belonging to a DosR-like dormancy regulon were abundant in S. Coelicolor, whereas tellurium resistance proteins were abundant in S. lividans. This indicated that the strains reacted differently to O-2 limitation. Proteins belonging to the CDA, RED, and ACT pathways, usually highly expressed in S. Coelicolor, were not detected under these Conditions, whereas proteins of siderophores, 5-hydroxyectoine, and terpenoid biosynthetic pathways were present.
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Repression of Antibiotic Production and Sporulation in Streptomyces Coelicolor by Overexpression of a TetR Family Transcriptional Regulator
Applied and Environmental Microbiology, 2010Co-Authors: Nicolas Seghezzi, Catherine Esnault, Marie-jöelle VirolleAbstract:The overexpression of a regulatory gene of the TetR family (SCO3201) originating either from Streptomyces lividans or from Streptomyces Coelicolor was shown to strongly repress antibiotic production (calcium-dependent antibiotic [CDA], undecylprodigiosin [RED], and actinorhodin [ACT]) of S. Coelicolor and of the ppk mutant strain of S. lividans. Curiously, the overexpression of this gene also had a strong inhibitory effect on the sporulation process of S. Coelicolor but not on that of S. lividans. SCO3201 was shown to negatively regulate its own transcription, and its DNA binding motif was found to overlap its −35 promoter sequence. The interruption of this gene in S. lividans or S. Coelicolor did not lead to any obvious phenotypes, indicating that when overexpressed SCO3201 likely controls the expression of target genes of other TetR regulators involved in the regulation of the metabolic and morphological differentiation process in S. Coelicolor. The direct and functional interaction of SCO3201 with the promoter region of scbA, a gene under the positive control of the TetR-like regulator, ScbR, was indeed demonstrated by in vitro as well as in vivo approaches.
Monica Chander - One of the best experts on this subject based on the ideXlab platform.
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RNA-Seq Analysis Reveals a Six-Gene SoxR Regulon in Streptomyces Coelicolor
PloS one, 2014Co-Authors: Nawar Naseer, Joshua A. Shapiro, Monica ChanderAbstract:The redox-regulated transcription factor SoxR is conserved in diverse bacteria, but emerging studies suggest that this protein plays distinct physiological roles in different bacteria. SoxR regulates a global oxidative stress response (involving >100 genes) against exogenous redox-cycling drugs in Escherichia coli and related enterics. In the antibiotic producers Streptomyces Coelicolor and Pseudomonas aeruginosa, however, SoxR regulates a smaller number of genes that encode membrane transporters and proteins with homology to antibiotic-tailoring enzymes. In both S. Coelicolor and P. aeruginosa, SoxR-regulated genes are expressed in stationary phase during the production of endogenously-produced redox-active antibiotics. These observations suggest that SoxR evolved to sense endogenous secondary metabolites and activate machinery to process and transport them in antibiotic-producing bacteria. Previous bioinformatics analysis that searched the genome for SoxR-binding sites in putative promoters defined a five-gene SoxR regulon in S. Coelicolor including an ABC transporter, two oxidoreductases, a monooxygenase and an epimerase/dehydratase. Since this in silico screen may have missed potential SoxR-targets, we conducted a whole genome transcriptome comparison of wild type S. Coelicolor and a soxR-deficient mutant in stationary phase using RNA-Seq. Our analysis revealed a sixth SoxR-regulated gene in S. Coelicolor that encodes a putative quinone oxidoreductase. Knowledge of the full complement of genes regulated by SoxR will facilitate studies to elucidate the function of this regulatory molecule in antibiotic producers.
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Expression of the Streptomyces Coelicolor SoxR Regulon Is Intimately Linked with Actinorhodin Production
Journal of Bacteriology, 2010Co-Authors: Rica Dela Cruz, Yang Gao, Sahitya Penumetcha, Rebecca Sheplock, Katherine Weng, Monica ChanderAbstract:The [2Fe-2S]-containing transcription factor SoxR is conserved in diverse bacteria. SoxR is traditionally known as the regulator of a global oxidative stress response in Escherichia coli, but recent studies suggest that this function may be restricted to enteric bacteria. In the vast majority of nonenterics, SoxR is predicted to mediate a response to endogenously produced redox-active metabolites. We have examined the regulation and function of the SoxR regulon in the model antibiotic-producing filamentous bacterium Streptomyces Coelicolor. Unlike the E. coli soxR deletion mutant, the S. Coelicolor equivalent is not hypersensitive to oxidants, indicating that SoxR does not potentiate antioxidant defense in the latter. SoxR regulates five genes in S. Coelicolor, including those encoding a putative ABC transporter, two oxidoreductases, a monooxygenase, and a possible NAD-dependent epimerase/dehydratase. Expression of these genes depends on the production of the benzochromanequinone antibiotic actinorhodin and requires intact [2Fe-2S] clusters in SoxR. These data indicate that actinorhodin, or a redox-active precursor, modulates SoxR activity in S. Coelicolor to stimulate the production of a membrane transporter and proteins with homology to actinorhodin-tailoring enzymes. While the role of SoxR in S. Coelicolor remains under investigation, these studies support the notion that SoxR has been adapted to perform distinct physiological functions to serve the needs of organisms that occupy different ecological niches and face different environmental challenges.
Wolfgang Wohlleben - One of the best experts on this subject based on the ideXlab platform.
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The PII protein GlnK is a pleiotropic regulator for morphological differentiation and secondary metabolism in Streptomyces Coelicolor
Applied microbiology and biotechnology, 2011Co-Authors: Eva Waldvogel, Alexander Herbig, Florian Battke, Rafat Amin, Merle Nentwich, Kay Nieselt, Trond E. Ellingsen, Alexander Wentzel, David A. Hodgson, Wolfgang WohllebenAbstract:GlnK is an important nitrogen sensor protein in Streptomyces Coelicolor. Deletion of glnK results in a medium-dependent failure of aerial mycelium and spore formation and loss of antibiotic production. Thus, GlnK is not only a regulator of nitrogen metabolism but also of morphological differentiation and secondary metabolite production. Through a comparative transcriptomic approach between the S. Coelicolor wild-type and a S. Coelicolor glnK mutant strain, 142 genes were identified that are differentially regulated in both strains. Among these are genes of the ram and rag operon, which are involved in S. Coelicolor morphogenesis, as well as genes involved in gas vesicle biosynthesis and ectoine biosynthesis. Surprisingly, no relevant nitrogen genes were found to be differentially regulated, revealing that GlnK is not an important nitrogen sensor under the tested conditions.
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Proteomic analysis of the GlnR-mediated response to nitrogen limitation in Streptomyces Coelicolor M145.
Applied microbiology and biotechnology, 2011Co-Authors: Yvonne Tiffert, Mirita Franz-wachtel, Claudia Fladerer, Alfred Nordheim, Jens Reuther, Wolfgang Wohlleben, Yvonne MastAbstract:GlnR is the global regulator of nitrogen assimilation in Streptomyces Coelicolor M145 and other actinobacteria. Two-dimensional polyacrylamide gel electrophoresis analyses were performed to identify new GlnR target genes by proteomic comparison of wild-type S. Coelicolor M145 and a ΔglnR mutant. Fifty proteins were found to be differentially regulated between S. Coelicolor M145 and the ΔglnR mutant. These spots were identified by nanoHPLC–ESI-MS/MS and classified according to their cellular role. Most of the identified proteins are involved in amino acid biosynthesis and in carbon metabolism, demonstrating that the role of GlnR is not restricted to nitrogen metabolism. Thus, GlnR is supposed to play an important role in the global metabolic control of S. Coelicolor M145.
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the streptomyces Coelicolor glnr regulon identification of new glnr targets and evidence for a central role of glnr in nitrogen metabolism in actinomycetes
Molecular Microbiology, 2008Co-Authors: Yvonne Tiffert, Wolfgang Wohlleben, Petra Supra, Reinhild Wurm, Rolf Wagner, Jens ReutherAbstract:Streptomyces Coelicolor GlnR is a global regulator that controls genes involved in nitrogen metabolism. By genomic screening 10 new GlnR targets were identified, including enzymes for ammonium assimilation (glnII, gdhA), nitrite reduction (nirB), urea cleavage (ureA) and a number of biochemically uncharacterized proteins (SCO0255, SCO0888, SCO2195, SCO2400, SCO2404, SCO7155). For the GlnR regulon, a GlnR binding site which comprises the sequence gTnAc-n(6)-GaAAc-n(6)-GtnAC-n(6)-GAAAc-n(6) has been found. Reverse transcription analysis of S. Coelicolor and the S. Coelicolor glnR mutant revealed that GlnR activates or represses the expression of its target genes. Furthermore, glnR expression itself was shown to be nitrogen-dependent. Physiological studies of S. Coelicolor and the S. Coelicolor glnR mutant with ammonium and nitrate as the sole nitrogen source revealed that GlnR is not only involved in ammonium assimilation but also in ammonium supply. blast analysis demonstrated that GlnR-homologous proteins are present in different actinomycetes containing the glnA gene with the conserved GlnR binding site. By DNA binding studies, it was furthermore demonstrated that S. Coelicolor GlnR is able to interact with these glnA upstream regions. We therefore suggest that GlnR-mediated regulation is not restricted to Streptomyces but constitutes a regulon conserved in many actinomycetes.
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Nitrogen metabolism in Streptomyces Coelicolor A3(2): modification of glutamine synthetase I by an adenylyltransferase
Microbiology, 1999Co-Authors: D. Fink, Wolfgang Wohlleben, D Falke, A. EngelsAbstract:An internal adenylyltransferase gene (glnE) fragment from Streptomyces Coelicolor was amplified using heterologous PCR primers derived from consensus motifs. The sequence had significant similarity to bacterial glnE genes, and included a motif typical of the C-terminal adenylyltransferase domain of glnE. glnE from S. Coelicolor lies on the Asel-C fragment of the chromosome and is localized near glnA (encoding glutamine synthetase I, GSI) and glnII (encoding GSII). To analyse the function of glnE in S. Coelicolor, glnE (S. Coelicolor E4) and glnA (S. Coelicolor HT107) gene replacement mutants were constructed. The GSI activity of the glnE mutant was not down-regulated after an ammonium shock. However, the GSI activity of the wild-type cells decreased to 60% of the original activity. The glnA mutant is not glutamine auxotrophic, but in the gamma-glutamyltransferase assay no GSI activity was detected in unshifted and shifted HT107 cells. By snake venom phosphodiesterase treatment the GSI activity in the wild-type can be reconstituted, whereas no alteration is observed in the E4 mutant. Additionally, the loss of short-term GSI regulation in the E4 mutant was accompanied by an increased glutamine:glutamate ratio.
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Increased yield of a lysozyme after self-cloning of the gene in Streptomyces Coelicolor "Müller".
Applied microbiology and biotechnology, 1991Co-Authors: Barbara Bräu, Wolfgang Wohlleben, R. Hilgenfeld, Merten Schlingmann, Marquardt Ruediger, Elli Birr, Karin Aufderheide, Alfred PühlerAbstract:Streptomyces Coelicolor “Muller” DSM3030 excretes a lysozyme comprising both β-1,4-N-acetyl-and β-1,4-N,6-O-diacetyl muramidase activities. The lysozyme is named Cellosyl. Gene libraries have been established using genomic DNA from the wild-type strain, S. Coelicolor DSM3030, and from an overproducing mutant, S. Coelicolor HP1, which exhibits about a twofold increase in lysozome production. The lysozyme-encoding genes (cel) from both strains were detected by oligodeoxynucleotide hybridization. The nucleotide sequence of the cel genes isolated from both strains was shown to be identical. The different levels of lysozyme production could not be correlated with any mutations at the cel gene locus. The cel gene isolated from the wild-type strain could not be expressed in some other species of Streptomyces. However, self-cloning of the cel gene into S. Coelicolor DSM3030 and HP1 resulted in a 2.5-fold increase in lysozyme production.
Sergio Sánchez - One of the best experts on this subject based on the ideXlab platform.
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Cloning and expression of the sco2127 gene from Streptomyces Coelicolor M145.
Journal of industrial microbiology & biotechnology, 2009Co-Authors: Adán Chávez, Romina Rodríguez-sanoja, Yolanda García-huante, Elizabeth Langley, Beatriz Ruiz, Sergio SánchezAbstract:It is known that Streptomyces peucetius var. caesius mutants resistant to 2-deoxyglucose (DogR) exhibit glucose transport deficiency, low glucose kinase (Glk) activity and insensitivity to carbon catabolite repression (CCR). This phenotype can be pleiotropically complemented by a 576-bp gene encoding SCO2127 from Streptomyces Coelicolor, suggesting the participation of this protein in the CCR process. In the present work, the sco2127 region was subcloned into pQE30 and its transcription product (SCO2127-His6) overexpressed. This procedure allowed purification of SCO2127 (with a Ni-sepharose resin) and production of polyclonal antibodies. In western blot assays, the antibodies gave a positive reaction against protein extracts from both S. Coelicolor and S. peucetius var. caesius, appearing as a single band of 34 kDa. No protein was detected using extracts from a S. Coelicolor mutant lacking the sco2127 gene (Δsco2127). In agreement with its possible involvement in the CCR process, SCO2127 was detected during the logarithmic growth phase of S. Coelicolor grown in minimal medium supplemented with 50 and 100 mM glucose. In addition, when 50 mM glucose was utilized, SCO2127 and residual glucose concentration simultaneously decreased at later stages of the microbial growth.
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Biochemical characterization of the glucose kinase from Streptomyces Coelicolor compared to Streptomyces peucetius var. caesius
Research in microbiology, 2004Co-Authors: Iveta Imriskova, Romina Rodríguez-sanoja, Elizabeth Langley, Roberto Arreguín-espinosa, Silvia Guzmán, Sergio SánchezAbstract:Glucose kinase (Glk) from Streptomyces Coelicolor was purified, characterized biochemically and kinetically, and compared to Glk from Streptomyces peucetius var. caesius. In both Streptomyces, 96% of the enzyme activity was detected in the cytosolic fraction. The use of a Glk activity gel showed no isoforms of the enzyme in extracts from these microorganisms. The purified S. Coelicolor Glk was stable in its tetrameric form, unlike the purified enzyme from S. peucetius var. caesius which easily dissociated into dimers. Tetramer dissociation was prevented by 100 mM d-glucose; however, this effect was not observed with other sugars. The optimum pH and the pI are practically identical for both enzymes. Maximum activity was found at a lower temperature for S. Coelicolor Glk (33 compared to 42 degrees C) and its activity was apparently less stable at higher temperatures. Its activation energy was also 40% lower than that of S. peucetius var. caesius. The kinetic mechanism appears to follow a rapid equilibrium-ordered Bi-Bi sequential mechanism in both microbial enzymes, where Glk first binds glucose and then the MgATP(2-) complex, to form a ternary complex (enzyme d-glucose-MgATP(-2)). The Km values for D-glucose and MgATP(2-) were 1.4, 0.5 mM and 1.6, 0.8 mM for the S. Coelicolor and S. peucetius var. caesius Glks, respectively. However Vmax of S. Coelicolor Glk was higher. In conclusion, the S. Coelicolor Glk showed a more stable tetrameric form with better affinity for its substrates and higher Vmax, suggesting greater catalytic efficiency.
Elizabeth Langley - One of the best experts on this subject based on the ideXlab platform.
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Interaction of SCO2127 with BldKB and its possible connection to carbon catabolite regulation of morphological differentiation in Streptomyces Coelicolor
Applied Microbiology and Biotechnology, 2011Co-Authors: Adán Chávez, Angela Forero, Mauricio Sánchez, Romina Rodríguez-sanoja, Guillermo Mendoza-hernández, Luis Servín-gonzalez, Brenda Sánchez, Yolanda García-huante, Diana Rocha, Elizabeth LangleyAbstract:In Streptomyces Coelicolor , the sco2127 gene is located upstream of the gene encoding for glucose kinase. This region restores sensitivity to carbon catabolite repression (CCR) of Streptomyces peucetius var. caesius mutants, resistant to 2-deoxyglucose (Dog^R). In order to search for the possible mechanisms behind this effect, sco2127 was overexpressed and purified for protein–protein interaction studies. SCO2127 was detected during the late growth phase of S. Coelicolor grown in a complex media supplemented with 100 mM glucose. Pull-down assays using crude extracts from S. Coelicolor grown in the same media, followed by far-western blotting, allowed detection of two proteins bound to SCO2127. The proteins were identified by MALDI-TOF mass spectrometry as SCO5113 and SCO2582. SCO5113 (BldKB) is a lipoprotein ABC-type permease (∼66 kDa) involved in mycelium differentiation by allowing the transport of the morphogenic oligopeptide Bld261. SCO2582, is a putative membrane metalloendopeptidase (∼44 kDa) of unknown function. In agreement with the possible role of SCO2127 in mycelium differentiation, delayed aerial mycelium septation and sporulation was observed when S. Coelicolor A3(2) was grown in the presence of elevated glucose concentrations (100 mM), an effect not seen in a Δ- sco2127 mutant derived from it. We speculate that SCO2127 might represent a key factor in CCR of mycelium differentiation by interacting with BldKB.
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Cloning and expression of the sco2127 gene from Streptomyces Coelicolor M145.
Journal of industrial microbiology & biotechnology, 2009Co-Authors: Adán Chávez, Romina Rodríguez-sanoja, Yolanda García-huante, Elizabeth Langley, Beatriz Ruiz, Sergio SánchezAbstract:It is known that Streptomyces peucetius var. caesius mutants resistant to 2-deoxyglucose (DogR) exhibit glucose transport deficiency, low glucose kinase (Glk) activity and insensitivity to carbon catabolite repression (CCR). This phenotype can be pleiotropically complemented by a 576-bp gene encoding SCO2127 from Streptomyces Coelicolor, suggesting the participation of this protein in the CCR process. In the present work, the sco2127 region was subcloned into pQE30 and its transcription product (SCO2127-His6) overexpressed. This procedure allowed purification of SCO2127 (with a Ni-sepharose resin) and production of polyclonal antibodies. In western blot assays, the antibodies gave a positive reaction against protein extracts from both S. Coelicolor and S. peucetius var. caesius, appearing as a single band of 34 kDa. No protein was detected using extracts from a S. Coelicolor mutant lacking the sco2127 gene (Δsco2127). In agreement with its possible involvement in the CCR process, SCO2127 was detected during the logarithmic growth phase of S. Coelicolor grown in minimal medium supplemented with 50 and 100 mM glucose. In addition, when 50 mM glucose was utilized, SCO2127 and residual glucose concentration simultaneously decreased at later stages of the microbial growth.
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Biochemical characterization of the glucose kinase from Streptomyces Coelicolor compared to Streptomyces peucetius var. caesius
Research in microbiology, 2004Co-Authors: Iveta Imriskova, Romina Rodríguez-sanoja, Elizabeth Langley, Roberto Arreguín-espinosa, Silvia Guzmán, Sergio SánchezAbstract:Glucose kinase (Glk) from Streptomyces Coelicolor was purified, characterized biochemically and kinetically, and compared to Glk from Streptomyces peucetius var. caesius. In both Streptomyces, 96% of the enzyme activity was detected in the cytosolic fraction. The use of a Glk activity gel showed no isoforms of the enzyme in extracts from these microorganisms. The purified S. Coelicolor Glk was stable in its tetrameric form, unlike the purified enzyme from S. peucetius var. caesius which easily dissociated into dimers. Tetramer dissociation was prevented by 100 mM d-glucose; however, this effect was not observed with other sugars. The optimum pH and the pI are practically identical for both enzymes. Maximum activity was found at a lower temperature for S. Coelicolor Glk (33 compared to 42 degrees C) and its activity was apparently less stable at higher temperatures. Its activation energy was also 40% lower than that of S. peucetius var. caesius. The kinetic mechanism appears to follow a rapid equilibrium-ordered Bi-Bi sequential mechanism in both microbial enzymes, where Glk first binds glucose and then the MgATP(2-) complex, to form a ternary complex (enzyme d-glucose-MgATP(-2)). The Km values for D-glucose and MgATP(2-) were 1.4, 0.5 mM and 1.6, 0.8 mM for the S. Coelicolor and S. peucetius var. caesius Glks, respectively. However Vmax of S. Coelicolor Glk was higher. In conclusion, the S. Coelicolor Glk showed a more stable tetrameric form with better affinity for its substrates and higher Vmax, suggesting greater catalytic efficiency.