Biochemical characterization of recombinant phosphoglucose isomerase of Mycobacterium tuberculosis

Bacillus sphaericus cannot metabolize sugar since it lacks several of the enzymes necessary for glycolysis. Our results confirmed the presence of a glucokinase-encoding gene, glcK, and a phosphofructokinase-encoding gene, pfk, on the bacterial chromosome and expression of glucokinase during vegetative growth of B. sphaericus strains. However, no phosphoglucose isomerase gene (pgi) or phosphoglucose isomerase enzyme activity was detected in these strains. Furthermore, one glcK open reading frame was cloned from B. sphaericus strain C3-41 and then expressed in Escherichia coli. Biochemical analysis revealed that this gene encoded a protein with a molecular mass of 33 kDa and that the purified recombinant glucokinase had K m values of 0.52 and 0.31 mM for ATP and glucose, respectively. It has been proved that this ATP-dependent glucokinase can also phosphorylate fructose and mannose, and sequence alignment of the glcK gene indicated that it belongs to the ROK protein family. It is postulated that the absence of the phosphoglucose isomerase-encoding gene pgi in B. sphaericus might be one of the reasons for the inability of this bacterium to metabolize carbohydrates. Our findings provide additional data that further elucidate the specific metabolic pathway and could be used for genetic improvement of B. sphaericus.Bacillus sphaericus is an aerobic, mesophilic, spore-forming bacterium with terminal swollen sporangia and spherical spores. As a consequence of the specific toxicity to mosquito larvae of binary toxin (Bin) and mosquitocidal toxins (Mtxs) produced during the sporulation and vegetative stages, respectively, some toxic strains have been widely used for many years as biopesticides in the field in mosquito control programs.B. sphaericus can metabolize a wide variety of organic compounds and amino acids, but it is unable to use hexoses and pentoses as unique carbon sources (20). The specific metabolic limitation of B. sphaericus hampers its potential industrial development due to the high costs of the proteinaceous media used for toxin production compared to the costs of alternative media based on starch or molasses. Consequently, studies elucidating the whole metabolic potential of B. sphaericus are essential for genetic improvement of this bacterium.Previous research indicated that the inability of B. sphaericus to metabolize carbohydrates could be attributed to its inability to transport glucose or sucrose into the cell (24) and the absence of key enzyme activities in the Embden-Meyerhof-Parnas (EMP) (glucokinase, glucose-6-phosphate isomerase, 6-phosphofructokinase), hexose monophosphate pathway (phosphogluconate dehydratase), and Entner-Doudoroff (6-phospho-2-keto-3-deoxyglyconate aldolase) pathways (20). Recent studies revealed that B. sphaericus strain 2362 has a glucose transport system (2) and that it also has 6-phosphofructokinase activity; thus, it could use Nacetylglucosamine as a sole carbon source (3). However, there are no data available about the presence of gene and enzyme activities of glucose...

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“…were assayed separately using the methods described by Mathur et al (15) and Alice et al (3), respectively.…”

Section: Bacterialmentioning

confidence: 99%

Molecular Characterization of a Glucokinase with Broad Hexose Specificity from Bacillus sphaericus Strain C3-41

Han

Liu

et al. 2007

Appl Environ Microbiol

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“…From this aspect, PGI, an enzyme essential for the survival of Myc. tbc, may be treated as a potential target of compounds with antituberculosis activity 5,7 . Secondly, as mentioned in the Introduction section, the characterized biocatalyst is known to be responsible for the formation of arabinogalactan, a key component of mycobacterial cell wall.…”

Section: Resultsmentioning

confidence: 99%

“…To our best knowledge, in contrast to 6-phosphogluconate, which has been listed as one of the most important inhibitors of the PGI activity 4,7 , the influence of PEP on the activity of mycobacterial PGI had never been examined before. PEP was reported to be an effective inhibitor of PGIs from Bacillus caldotenax 55 and E. coli 56 Ogawa et al 56 found that PEP exhibited a competitive type of inhibition of E. coli PGI with respect to fru-6P.…”

Section: Zymomonas Mobilismentioning

confidence: 99%

“…Apart from its enzymatic function, this protein has a number of other ''moonlighting'' functions 5 . In mycobacteria, PGI is engaged through glucose-6P with L-rhamnose and is involved in the biosynthesis of the galactan residue of arabinogalactan, the components of the cell wall of these microorganisms [6][7][8] . The major application of bioautography is screening of the antimicrobial and antifungal properties of various plant samples with Thin Layer Chromatography (TLC)-plates 9 .…”

Section: Introductionmentioning

confidence: 99%

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Establishment of an effective TLC bioautographic method for the detection ofMycobacterium tuberculosisH37Ra phosphoglucose isomerase inhibition by phosphoenolpyruvate

Paradowska

Polak

Chomicki

et al. 2016

Journal of Enzyme Inhibition and Medicinal Chemistry

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A bioautographic assay based on thin layer chromatography was developed for phosphoenolpyruvate (PEP) detecting as a known but rarely studied inhibitor of phosphoglucose isomerase (PGI). The protocol with NADP + /NBT/PMS (b-nicotinamide adenine dinucleotide phosphate/nitrotetrazolium blue chloride/phenazine methosulfate) staining was capable of detecting Mycobacterium tuberculosis H 37 Ra PGI inhibition using PEP. According to this method, visibly brighter spots (zones) against purple background are observed in the area of inhibition of the above-mentioned enzyme activity. The detection limit for PEP as an inhibitor of Mycobacterium tuberculosis H 37 Ra PGI was 226 mg per spot/zone. Noteworthy is that we are the first authors to have successfully used a bioautographic assay to detect Mycobacterium tuberculosis H 37 Ra PGI inhibition by PEP.

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“…Considering that glucose is a preferred source of carbon and energy for many bacteria, the transport of this sugar into the cell and its subsequent phosphorylation is related to global gene regulation, and may also influence pathogenicity [Deol et al, 2005;Mathur et al, 2005]. M. tuberculosis virulence is correlated with a shift from a strict aerobic respiratory mode to anaerobic metabolism.…”

Section: Introductionmentioning

confidence: 99%

A Glucose Kinase from <i>Mycobacterium smegmatis</i>

Pimentel-Schmitt

Thomae²,

Amon

et al. 2006

Microb Physiol

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Carbon metabolism and regulation is poorly understood in mycobacteria, a genus that includes some major pathogenic species like Mycobacterium tuberculosis and Mycobacterium leprae. Here, we report the identification of a glucose kinase from Mycobacterium smegmatis. This enzyme serves in glucose metabolism and global carbon catabolite repression in the related actinomycete Streptomyces coelicolor. The gene, msmeg1356 (glkA), was found by means of in silico screening. It was shown that it occurs in the same genetic context in all so far sequenced mycobacterial species, where it is located in a putative tricistronic operon together with a glycosyl hydrolase and a putative malonyl-CoA transacylase. Heterologous expression of glkA in an Escherichia coli glucose kinase mutant led to the restoration of glucose growth, which provided in vivo evidence for glucose kinase function. GlkA^Msm was subsequently overproduced in order to study its enzymatic features. We found that it can form a dimer and that it efficiently phosphorylates glucose at the expense of ATP. The affinity constant for glucose was with 9 mM about eight times higher and the velocity was about tenfold slower when compared to the parallel measured glucose kinase of S. coelicolor. Both enzymes showed similar substrate specificity, which consists in an ATP-dependent phosphorylation of glucose and no, or very inefficient, phosphorylation of the glucose analogues 2-deoxyglucose and methyl α-glucoside. Hence, our data provide a basis for studying the role of mycobacterial glucose kinase in vivo to unravel possible catalytic and regulatory functions.

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Biochemical characterization of recombinant phosphoglucose isomerase of Mycobacterium tuberculosis

Cited by 15 publications

References 31 publications

Molecular Characterization of a Glucokinase with Broad Hexose Specificity from Bacillus sphaericus Strain C3-41

Molecular Characterization of a Glucokinase with Broad Hexose Specificity from Bacillus sphaericus Strain C3-41

Establishment of an effective TLC bioautographic method for the detection ofMycobacterium tuberculosisH37Ra phosphoglucose isomerase inhibition by phosphoenolpyruvate

A Glucose Kinase from <i>Mycobacterium smegmatis</i>

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