The transglycosylation reaction of cyclodextrin glucanotransferase is operated by a Ping‐Pong mechanism

Nakamura, Akira; Haga, Keiko; Yamane, Kunio

doi:10.1016/0014-5793(94)80631-4

Cited by 79 publications

(56 citation statements)

References 26 publications

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“…This replacement conformed to the observation of aromatic residues at that position in natural glycoside transferases (like CGTases), as well as in a conserved region among maltogenic glycoside hydrolases (10) and in saccharifying amylases of fungal origin (54). It has been widely accepted that an aromatic residue at position 286 (B. licheniformis number) is a key condition for transglycosylation (34,40,45,50,54). An interesting observation is the conservation of a phenylalanine residue at position 277 in AmyA (equivalent to position 286 in B. licheniformis and 289 in B. stearothermophilus ␣-amylases), in agreement with the importance of an aromatic residue at this position for transglycosylation activity.…”

Section: Discussionsupporting

confidence: 74%

“…The importance of aromatic residues in substrate binding at the active site of glycosidases through stacking interactions is well documented (7), and it has been proven that the introduction or substitution of these kinds of residues can modify the affinity of the protein at the different substrate subsites, affecting both the product profile and the activity of the enzyme (40,45,50,54).…”

mentioning

confidence: 99%

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Enhancement of the Alcoholytic Activity of α-Amylase AmyA fromThermotoga maritimaMSB8 (DSM 3109) by Site-Directed Mutagenesis

Damián-Almazo

Moreno

López-Munguı́a

et al. 2008

Appl Environ Microbiol

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AmyA, an ␣-amylase from the hyperthermophilic bacterium Thermotoga maritima, is able to hydrolyze internal ␣-1,4-glycosidic bonds in various ␣-glucans at 85°C as the optimal temperature. Like other glycoside hydrolases, AmyA also catalyzes transglycosylation reactions, particularly when oligosaccharides are used as substrates. It was found that when methanol or butanol was used as the nucleophile instead of water, AmyA was able to catalyze alcoholysis reactions. This capability has been evaluated in the past for some ␣-amylases, with the finding that only the saccharifying fungal amylases from Aspergillus niger and from Aspergillus oryzae present measurable alcoholysis activity (R. I. Santamaria, G. Del Rio, G. Saab, M. E. Rodriguez, X. Soberon, and A. Lopez, FEBS Lett. 452:346-350, 1999). In the present work, we found that AmyA generates larger quantities of alkyl glycosides than any amylase reported so far. In order to increase the alcoholytic activity observed in AmyA, several residues were identified and mutated based on previous analogous positions in amylases, defining the polarity and geometry of the active site. Replacement of residue His222 by glutamine generated an increase in the alkyl glucoside yield as a consequence of a higher alcoholysis/hydrolysis ratio. The same change in specificity was observed for the mutants H222E and H222D, but instability of these mutants toward alcohols decreased the yield of alkyl glucoside.␣-Amylases (EC 3.2.1.1) are retaining glycosidases that catalyze the hydrolysis of internal ␣-1,4-glycosidic bonds in starch through a double-displacement mechanism in which a covalent intermediate glycosyl enzyme is deglycosylated by water (43, 62). ␣-Amylases contain 5 to 11 subsites that bind glucose moieties (8, 51), with their numbers and relative affinities defining their product profiles (38). Like all retaining glycosidases, ␣-amylases can also catalyze transfer reactions, which are the result of employing molecules other than water as glucosyl acceptors, such as carbohydrates (transglycosylation reactions) or alcohols (alcoholysis reactions). When a highmolecular-weight alcohol is used as an acceptor, the product is an alkyl glycoside with surface tension activity properties that are important in several industrial applications. Therefore, the extremely laborious and inefficient chemical synthesis of alkyl glycosides presents an opportunity to develop enzymatic methods devoted to increasing the yields and specificities of these reactions.The feasibility of alcoholysis reactions using various exoglycosidases has been extensively investigated (references 57 and 65 and references therein), and although efficient processes have been developed using activated substrates, such as pnitrophenyl-glucoside or p-nitrophenyl-galactoside, with ␣-and ␤-glucosidases and galactosidases, the use of a readily available substrate, such as starch or amylodextrins, could prove attractive if efficient reactions employing ␣-amylases are developed.For a given degree of starch depolymerization, endoa...

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Section: Discussionsupporting

confidence: 74%

mentioning

confidence: 99%

Enhancement of the Alcoholytic Activity of α-Amylase AmyA fromThermotoga maritimaMSB8 (DSM 3109) by Site-Directed Mutagenesis

Damián-Almazo

Moreno

López-Munguı́a

et al. 2008

Appl Environ Microbiol

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“…It had been demonstrated for CGTase that the transglycosylation reaction (and, therefore, most likely also the cyclization reaction) operates by a ping-pong bi-bi mechanism [27]. The enzyme binds a linear A-glucan of undefined length and splits it between subsites 1 and 1′.…”

Section: Induced-fitmentioning

confidence: 99%

“…Their central importance was established by site-directed mutagenesis in conjunction with crystallographic studies [18Ϫ25]. All family members retain the configuration at the anomeric carbon of the scissile bond [26,27].…”

mentioning

confidence: 99%

Substrate binding to a cyclodextrin glycosyltransferase and mutations increasing the γ‐cyclodextrin production

Parsiegla

Schmidt

Schulz

1998

European Journal of Biochemistry

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Bacterial cyclodextrin glycosyltransferases use starch to produce cyclic maltooligosaccharides (cyclodextrins) which are of interest in various applications. The cyclization reaction gives rise to a spectrum of ring sizes consisting of predominantly six to eight glucosyl units. Using the enzyme from Bacillus circulans strain no. 8, binding studies have been performed with several substrates and analogues. The observed binding modes differ in detail, but agree in general with data on homologous enzymes. Based on these binding studies, two mutations were designed that changed the production spectrum from the predominant product β‐cyclodextrin of the wild‐type enzyme towards γ‐cyclodextrin, which is of practical interest because it is rare and can encapsulate larger nonpolar compounds.

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“…Therefore, these cyclomaltodextrins are widely used in the pharmaceutical, food, and cosmetic industries (21,24). Since each cyclomaltodextrin has a distinct spectrum for guest molecules (13), extensive studies have been carried out to understand the mechanism of the cyclization reaction (1,16,17,22,25,(33)(34)(35) and to find or engineer CGTases to produce specific types of cyclomaltodextrin (18,23,(36)(37)(38).…”

mentioning

confidence: 99%

Comparative Study of the Cyclization Reactions of Three Bacterial Cyclomaltodextrin Glucanotransferases

Terada

Sanbe

Takaha

et al. 2001

Appl Environ Microbiol

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The actions of cyclomaltodextrin glucanotransferases (CGTase; EC 2.4.1.19) from alkalophilic Bacillus sp. strain A2-5a (A2-5a CGTase), Bacillus macerans (Bmac CGTase), and Bacillus stearothermophilus (Bste CGTase) on amylose were investigated. All three enzymes produced large cyclic ␣-1,4-glucans (cycloamyloses) at the early stage of the reaction, but these were subsequently converted into smaller cycloamyloses. However, the rates of this conversion differed among the three enzymes. The product specificity of each CGTase in the cyclization reaction was determined by measuring the amount of each cycloamylose from CD6 to CD31 (CDn, a cycloamylose with a degree of polymerization of n). A2-5a CGTase produced 10 times more CD7, while Bmac CGTase produced 34 times more CD6 than other cycloamyloses. Bste CGTase produced 12 and 3 times more CD6 and CD7 than other cycloamyloses, respectively. The substrate specificities of the linearization reactions of CD6, CD7, CD8, and larger cycloamyloses (a mixture of CD22 to CD50) were investigated, and we found that CD7 and CD8 are extremely poor substrates for both hydrolytic and transglycosidic linearization (coupling) reactions while larger cycloamyloses are linearized at a much higher rate. By repeating these cyclization and linearization reactions, the larger cycloamyloses initially produced are converted into smaller cycloamyloses and finally into mainly CD6, CD7, and CD8. These three enzymes also differ in their hydrolytic activities, which seem to accelerate the conversion of larger cycloamyloses into smaller cycloamyloses.

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The transglycosylation reaction of cyclodextrin glucanotransferase is operated by a Ping‐Pong mechanism

Cited by 79 publications

References 26 publications

Enhancement of the Alcoholytic Activity of α-Amylase AmyA fromThermotoga maritimaMSB8 (DSM 3109) by Site-Directed Mutagenesis

Enhancement of the Alcoholytic Activity of α-Amylase AmyA fromThermotoga maritimaMSB8 (DSM 3109) by Site-Directed Mutagenesis

Substrate binding to a cyclodextrin glycosyltransferase and mutations increasing the γ‐cyclodextrin production

Comparative Study of the Cyclization Reactions of Three Bacterial Cyclomaltodextrin Glucanotransferases

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