Mechanism of thermal denaturation of maltodextrin phosphorylase from Escherichia coli

Griessler, Richard; D’Auria, Sabato; Schinzel, Reinhard; Tanfani, Fabio; Nidetzky, Bernd

doi:10.1042/bj3460255

Cited by 5 publications

(1 citation statement)

References 40 publications

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“…The protein pellet obtained after centrifugation (10 000 g for 30 min) was dissolved in a small volume of 300 m m potassium phosphate buffer, pH 7.0, and incubated at 60 °C for 40 min. Note that heat treatment inactivates any remaining endogenous E. coli maltodextrin phosphorylase [19]. After centrifugation and concentration using 30‐kDa Microsep tubes (Pall Filtron), further purification was carried out by size exclusion chromatography on Superose 12 Prep Grade (Amersham Pharmacia Biotech; 16 mm diameter, 140 mL) equilibrated with 50 m m phosphate buffer, pH 7.0, containing 0.2 m NaCl.…”

Section: Purification and Characterization Of Recombinant Stp And Mutmentioning

confidence: 99%

Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae

Griessler

Schwarz

Mucha³

et al. 2003

European Journal of Biochemistry

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Glycogen phosphorylases (GPs) constitute a family of widely spread catabolic a1,4-glucosyltransferases that are active as dimers of two identical, pyridoxal 5¢-phosphatecontaining subunits. In GP from Corynebacterium callunae, physiological concentrations of phosphate are required to inhibit dissociation of protomers and cause a 100-fold increase in kinetic stability of the functional quarternary structure. To examine interactions involved in this large stabilization, we have cloned and sequenced the coding gene and have expressed fully active C. callunae GP in Escherichia coli. By comparing multiple sequence alignment to structurefunction assignments for regulated and nonregulated GPs that are stable in the absence of phosphate, we have scrutinized the primary structure of C. callunae enzyme for sequence changes possibly related to phosphate-dependent dimer stability. Location of Arg234, Arg236, and Arg242 within the predicted subunit-to-subunit contact region made these residues primary candidates for site-directed mutagenesis. Individual Arg fi Ala mutants were purified and characterized using time-dependent denaturation assays in urea and at 45°C. R234A and R242A are enzymatically active dimers and in the absence of added phosphate, they display a sixfold and fourfold greater kinetic stability of quarternary interactions than the wild-type, respectively. The stabilization by 10 mM of phosphate was, however, up to 20-fold greater in the wild-type than in the two mutants. The replacement of Arg236 by Ala was functionally silent under all conditions tested. Arg234 and Arg242 thus partially destabilize the C. callunae GP dimer structure, and phosphate binding causes a change of their tertiary or quartenary contacts, likely by an allosteric mechanism, which contributes to a reduced protomer dissociation rate.Keywords: interface; oxyanion; phosphate; stabilization; subunit dissociation.Glycogen phosphorylases (GPs) catalyse degradation of glycogen and structurally related reserve polysaccharides in the cytosol to provide energy via the branch point metabolite a-D-glucose-1-phosphate. All known GPs are functional homodimers composed of % 90-kDa subunits and require pyridoxal 5¢-phosphate (PLP) cofactor for activity [1][2][3][4][5][6][7]. Although a very low basal activity may be present in the holoenzyme protomer, quarternary interactions clearly determine physiological levels of phosphorylase activity and are a prerequisite for the regulatory properties of eukaryotic GPs [8][9][10]. Forces that stabilize the dimer structure of GP are therefore essential to optimal enzyme function under physiological boundary conditions. GPs are a/b proteins that display a twodomain fold in which the N-terminal domain and the C-terminal domain are separated by a catalytic site cleft. The structural elements that comprise the subunit-subunit interface are located in the N-terminal domain. The dimer contact regions of regulated and nonregulated GPs share structural similiarity overall, but differ on the molecular level [3][4][5][6][7...

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Section: Purification and Characterization Of Recombinant Stp And Mutmentioning

confidence: 99%

Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae

Griessler

Schwarz

Mucha³

et al. 2003

European Journal of Biochemistry

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Thermostable alpha-glucan phosphorylases: characteristics and industrial applications

Ubiparip

Beerens

Franceus

et al. 2018

Appl Microbiol Biotechnol

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α-Glucan phosphorylases (α-GPs) catalyze the reversible phosphorolysis of α-1,4-linked polysaccharides such as glycogen, starch, and maltodextrins, therefore playing a central role in the usage of storage polysaccharides. The discovery of these enzymes and their role in the course of catalytic conversion of glycogen was rewarded with the Nobel Prize in Physiology or Medicine in 1947. Nowadays, however, thermostable representatives attract special attention due to their vast potential in the enzymatic production of diverse carbohydrates and derivatives such as (functional) oligo- and (non-natural) polysaccharides, artificial starch, glycosides, and nucleotide sugars. One of the most recently explored utilizations of α-GPs is their role in the multi-enzymatic process of energy production stored in carbohydrate biobatteries. Regardless of their use, thermostable α-GPs offer significant advantages and facilitated bioprocess design due to their high operational temperatures. Here, we present an overview and comparison of up-to-date characterized thermostable α-GPs with a special focus on their reported biotechnological applications.

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Thermal denaturation pathway of starch phosphorylase from Corynebacterium callunae: Oxyanion binding provides the glue that efficiently stabilizes the dimer structure of the protein

et al. 2000

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Starch phosphorylase from Corynebacterium callunae is a dimeric protein in which each mol of 90 kDa subunit contains 1 mol pyridoxal 59-phosphate as an active-site cofactor. To determine the mechanism by which phosphate or sulfate ions bring about a greater than 500-fold stabilization against irreversible inactivation at elevated temperatures~Ն50 8C!, enzyme0oxyanion interactions and their role during thermal denaturation of phosphorylase have been studied. By binding to a protein site distinguishable from the catalytic site with dissociation constants of K sulfate ϭ 4.5 mM and K phosphate Ϸ 16 mM, dianionic oxyanions induce formation of a more compact structure of phosphorylase, manifested by~a! an increase by about 5% in the relative composition of the a-helical secondary structure,~b! reduced 1 H0 2 H exchange, and~c! protection of a cofactor fluorescence against quenching by iodide. Irreversible loss of enzyme activity is triggered by the release into solution of pyridoxal 59-phosphate, and results from subsequent intermolecular aggregation driven by hydrophobic interactions between phosphorylase subunits that display a temperature-dependent degree of melting of secondary structure. By specifically increasing the stability of the dimer structure of phosphorylasẽ probably due to tightened intersubunit contacts!, phosphate, and sulfate, this indirectly~1! preserves a functional active site up to Ϸ50 8C, and~2! stabilizes the covalent protein cofactor linkage up to Ϸ70 8C. The effect on thermostability shows a sigmoidal and saturatable dependence on the concentration of phosphate, with an apparent binding constant at 50 8C of Ϸ25 mM. The extra stability conferred by oxyanion-ligand binding to starch phosphorylase is expressed as a dramatic shift of the entire denaturation pathway to a Ϸ20 8C higher value on the temperature scale.Keywords: a-glucan phosphorylase; denaturation mechanism; oxyanion ligand; phosphate stabilization Unlike a great number of a-d-glycoside hydrolases, a-1,4-dglucan phosphorylases catalyze the degradation of a-1,4-d-glucan molecules by using phosphate rather than water as an acceptor of the transferred glucosyl moiety, as shown in Equation 1 where N is the degree of polymerization of the a-1,4-d-glucan.Therefore, interactions between the enzyme and the substrate phosphate play an essential role in the catalytic cycle of phosphorylase and contribute to binding energy in the ground state and the transition state of the reaction~Johnson et al

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Mechanism of thermal denaturation of maltodextrin phosphorylase from Escherichia coli

Cited by 5 publications

References 40 publications

Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae

Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae

Thermostable alpha-glucan phosphorylases: characteristics and industrial applications

Thermal denaturation pathway of starch phosphorylase from Corynebacterium callunae: Oxyanion binding provides the glue that efficiently stabilizes the dimer structure of the protein

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