Molecular characterization of <i>malQ</i>, the structural gene for the <i>Escherichia coli</i> enzyme amylomaltase

Pugsley, Anthony P.; Dubreuil, Christine

doi:10.1111/j.1365-2958.1988.tb00053.x

Cited by 62 publications

(38 citation statements)

References 26 publications

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“…The synthesis was done according to the method of Pajatsch et al (43) with alterations. Strain ST103 lacking MalQ, MalP, and MalZ was transformed with pCHAP113 harboring malQ under lac promoter control (47). The strain was grown in Luria broth and ampicillin (100 g/ml) in the absence of isopropyl-␤-D-thiogalactopyranoside (IPTG) (the strain did not contain the Lac repressor).…”

Section: Methodsmentioning

confidence: 99%

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The Maltodextrin System ofEscherichia coli: Metabolism and Transport

2005

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The maltose/maltodextrin regulon of Escherichia coli consists of 10 genes which encode a binding proteindependent ABC transporter and four enzymes acting on maltodextrins. All mal genes are controlled by MalT, a transcriptional activator that is exclusively activated by maltotriose. By the action of amylomaltase, we prepared uniformly labeled [ 14 C]maltodextrins from maltose up to maltoheptaose with identical specific radioactivities with respect to their glucosyl residues, which made it possible to quantitatively follow the rate of transport for each maltodextrin. Isogenic malQ mutants lacking maltodextrin phosphorylase (MalP) or maltodextrin glucosidase (MalZ) or both were constructed. The resulting in vivo pattern of maltodextrin metabolism was determined by analyzing accumulated [ 14 C]maltodextrins. MalP ؊ MalZ ؉ strains degraded all dextrins to maltose, whereas MalP ؉ MalZ ؊ strains degraded them to maltotriose. The labeled dextrins were used to measure the rate of transport in the absence of cytoplasmic metabolism. Irrespective of the length of the dextrin, the rates of transport at a submicromolar concentration were similar for the maltodextrins when the rate was calculated per glucosyl residue, suggesting a novel mode for substrate translocation. Strains lacking MalQ and maltose transacetylase were tested for their ability to accumulate maltose. At 1.8 nM external maltose, the ratio of internal to external maltose concentration under equilibrium conditions reached 10 6 to 1 but declined at higher external maltose concentrations. The maximal internal level of maltose at increasing external maltose concentrations was around 100 mM. A strain lacking malQ, malP, and malZ as well as glycogen synthesis and in which maltodextrins are not chemically altered could be induced by external maltose as well as by all other maltodextrins, demonstrating the role of transport per se for induction.

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Section: Methodsmentioning

confidence: 99%

“…MalQ is an amylomaltase (39,47,65,66), an obligatory glucanosyltransferase producing from any linear maltodextrin a mixture of maltodextrins plus glucose (44). Mutants lacking amylomaltase can no longer grow on maltose but can grow on maltodextrins with a chain length of four or more glucosyl residues (55).…”

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The Maltodextrin System ofEscherichia coli: Metabolism and Transport

2005

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“…furiosus, an obligately anaerobic and hyperthermophilic archaeon (Laderman et al, 1993 ; 65 % identity), and Dictyoglomus thermophilum, a Gram-negative, obligately anaerobic and extremely thermophilic bacterium (Fukusumi et al, 1988; 42% identity; Fig. 6), while the 7: litoralis 4-a-glucanotransferase showed a distant relationship to other 4-aglucanotransferases including potato D-enzyme (18 % identity ; Takaha et al, 1993) and E. coli amylomaltase (17% identity; Pugsley and Dubreuil, 1988). The 7: litoralis enzyme did not exhibit apparent sequence similarity with other sequence-determined enzymes.…”

Section: Ldxaakvwkfpiktlsqseagwdfiqqg----vsytmlfpiekel~ftvrfrel 659mentioning

confidence: 99%

4‐α‐Glucanotransferase from the Hyperthermophilic Archaeon Thermococcus Litoralis

Jeon

Taguchi

Sakai

et al. 1997

European Journal of Biochemistry

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4-a-Glucanotransferase was purified from cells of Thermococcus litoralis, a hyperthermophilic archaeon. The molecular mass of the enzyme was estimated to be approximately 87 kDa by gel filtration. The optimal temperature for its activity was 90 "C. The enzyme catalyzed the transglycosylation of maltooligosaccharides, yielding maltooligosaccharides of various lengths and glucose. When maltoheptaose was used as the substrate, glucoamylase-resistant and glucoamylase-sensitive saccharides were produced.On incubation of amylose with the 7: litoralis enzyme, glucoamylase-resistant but a-amylase-sensitive molecules were produced, but the amount of reducing sugar showed only slight increases. These results indicate that the 7: litoralis enzyme catalyzes not only intermolecular transglycosylation to produce linear a-1,4-glucan, but also intramolecular transglycosylation to produce cyclic a-1 ,4-glucan (cycloamylose), similarly to potato 4-a-glucanotransferase (called disproportionating enzyme). The gene encoding the 7: litoralis 4-a-glucanotransferase was cloned, sequenced and expressed in Escherichia coli. The nucleotide sequence of the gene encoded a 659-amino acid protein with a calculated molecular mass of 77 883 Da.The amino acid sequence of the 7: litoralis enzyme showed high similarity with those of a-amylases of Pyrococcus furiosus, a hyperthermophilic archaeon, and Dictyoglomus thermophilum, an extremely thermophilic bacterium, but little similarity with those of other known 4-a-glucanotransferases.Keywords: hyperthermophilic archaeon ; Thermococcus litoralis ; 4-a-glucanotransferase ; cyclic a-1,4-glucan; gene cloning.In recent years several hyperthermophilic archaea, capable of growing anaerobically, even at IOO"C, have been isolated from submarine volcanic areas (Fiala and Stetter, 1986;Kelly and Deming, 1988). One of the characteristics of these organisms is their ability to utilize complex saccharolytic substrates, such as starch and glycogen, as carbon and energy sources. Pyrococcus furiosus, a marine hyperthermophile that grows optimally at 98"C, is probably the best-characterized member of the thermophilic archaea. The metabolism of a variety of complex substrates by P: furiosus begins with their hydrolysis by a series of extracellular amylolytic enzymes exhibiting amylase and pullulanase activities (Koch et al., 1990;Brown et al., 1990). The ability to digest complex saccharides is also characteristic of Thermococcus litoralis, a hyperthermophilic marine archaeon with an optimal growth temperature of 85°C (Neuner et al., 1990). It appears to resemble R furiosus in many aspects of growth and metabolism, but it has not been well characterized.We searched for sugar-metabolizing enzymes produced by hyperthermophilic archaea, and in a cell-free extract of 7: litoCorrespondence to H . Matsuzawa,

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“…Thereby, MalQ forms larger maltodextrins and releases glucose (see Fig. 1) (Monod & Torriani, 1950;Pugsley & Dubreuil, 1988). Like the maltodextrins formed in the course of glycogen degradation, the MalQgenerated maltodextrins are degraded by either MalP or MalZ.…”

Section: Introductionmentioning

confidence: 99%

Roles of maltodextrin and glycogen phosphorylases in maltose utilization and glycogen metabolism in Corynebacterium glutamicum

2009

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Corynebacterium glutamicum transiently accumulates large amounts of glycogen, when cultivated on glucose and other sugars as a source of carbon and energy. Apart from the debranching enzyme GlgX, which is required for the formation of maltodextrins from glycogen, a-glucan phosphorylases were assumed to be involved in glycogen degradation, forming a-glucose 1-phosphate from glycogen and from maltodextrins. We show here that C. glutamicum in fact possesses two a-glucan phosphorylases, which act as a glycogen phosphorylase (GlgP) and as a maltodextrin phosphorylase (MalP). By chromosomal inactivation and subsequent analysis of the mutant, cg1479 was identified as the malP gene. The deletion mutant C. glutamicum DmalP completely lacked MalP activity and showed reduced intracellular glycogen degradation, confirming the proposed pathway for glycogen degradation in C. glutamicum via GlgP, GlgX and MalP. Surprisingly, the DmalP mutant showed impaired growth, reduced viability and altered cell morphology on maltose and accumulated much higher concentrations of glycogen and maltodextrins than the wild-type during growth on this substrate, suggesting an additional role of MalP in maltose metabolism of C. glutamicum. Further assessment of enzyme activities revealed the presence of 4-a-glucanotransferase (MalQ), glucokinase (Glk) and a-phosphoglucomutase (a-Pgm), and the absence of maltose hydrolase, maltose phosphorylase and b-Pgm, all three known to be involved in maltose utilization by Gram-positive bacteria. Based on these findings, we conclude that C. glutamicum metabolizes maltose via a pathway involving maltodextrin and glucose formation by MalQ, glucose phosphorylation by Glk and maltodextrin degradation via the reactions of MalP and a-Pgm, a pathway hitherto known to be present in Gram-negative rather than in Gram-positive bacteria. INTRODUCTIONCorynebacterium glutamicum is a Gram-positive soil bacterium employed in the production of various amino acids (Eggeling & Bott, 2005), and serves as a model organism for the mycolic acid-containing suborder Corynebacterianeae, which includes pathogenic species such as Corynebacterium diphtheriae, Mycobacterium tuberculosis and Mycobacterium leprae. The organism grows on a variety of mono-and disaccharides, alcohols and organic acids as single or combined sources of carbon and energy (Liebl, 2005). and transiently forms intracellular glycogen when cultivated on sugar substrates (Tzvetkov et al., 2003;. We recently showed that C. glutamicum forms maltodextrins in the course of glycogen degradation , and thus we speculate that glycogen is degraded in C. glutamicum by a pathway which proceeds similarly or identically to the one present in Escherichia coli. This pathway involves the interplay of six enzymes (Fig. 1). Glycogen phosphorylase (GlgP) cleaves a-1,4-glycosidic bonds at the non-reducing ends of glycogen and forms glucose 1-phosphate and phosphorylase-limited dextrins (pl-dextrins) (Dauvillee et al., 2005;Alonso-Casajus et al., 2006). Cleavage of the a-1,6-glycosidic bon...

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Molecular characterization of malQ, the structural gene for the Escherichia coli enzyme amylomaltase

Cited by 62 publications

References 26 publications

The Maltodextrin System ofEscherichia coli: Metabolism and Transport

The Maltodextrin System ofEscherichia coli: Metabolism and Transport

4‐α‐Glucanotransferase from the Hyperthermophilic Archaeon Thermococcus Litoralis

Roles of maltodextrin and glycogen phosphorylases in maltose utilization and glycogen metabolism in Corynebacterium glutamicum

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