Three histidine residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011: effects of the replacement on pH dependence and transition-state stabilization

Nakamura, Akira; Haga, Keiko; Yamane, Kunio

doi:10.1021/bi00077a015

Cited by 100 publications

(116 citation statements)

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“…Previously, structural and biochemical studies of CGTase had already provided evidence for the importance of His-233 in the ϩ1 acceptor binding subsite for catalysis (24). Here we concentrate on subsites ϩ2 and ϩ3, more distant from the catalytic site.…”

Section: Discussionmentioning

confidence: 97%

Hydrophobic Amino Acid Residues in the Acceptor Binding Site Are Main Determinants for Reaction Mechanism and Specificity of Cyclodextrin-glycosyltransferase

Veen¹,

Leemhuis²,

Kralj³

et al. 2001

Journal of Biological Chemistry

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Cyclodextrin-glycosyltransferases (CGTases) (EC 2.4.1.19) preferably catalyze transglycosylation reactions with glucosyl residues as acceptor, whereas the homologous ␣-amylases catalyze hydrolysis reactions using water as acceptor. This difference in reaction specificity is most likely caused by the acceptor binding site. To investigate this in detail we altered the acceptor site residues Lys-232, Phe-183, Phe-259, and Glu-264 of Bacillus circulans strain 251 CGTase using site-directed mutagenesis. Lys-232 is of general importance for catalysis, which appears to result mainly from stabilization of the conformation of the loop containing the catalytic nucleophile Asp-229 and His-233, a residue that has been implied in transition state stabilization. Glu-264 contributes to the disproportionation reaction only, where it is involved in initial binding of the (maltose) acceptor. Phe-183 and Phe-259 play important and distinct roles in the transglycosylation reactions catalyzed by CGTase. Mutation of Phe-183 affects especially the cyclization and coupling reactions, whereas Phe-259 is most important for the cyclization and disproportionation reactions. Moreover, the hydrophobisity of Phe-183 and Phe-259 limits the hydrolyzing activity of the enzyme. Hydrolysis can be enhanced by making these residues more polar, which concomitantly results in a lower transglycosylation activity. A double mutant was constructed that yielded an enzyme preferring hydrolysis over cyclization (15:1), whereas the wild type favors cyclization over hydrolysis (90:1).Cyclodextrin-glycosyltransferases (CGTase) 1 (EC 2.4.1.19) belong to the ␣ -amylase family (glycosyl hydrolase family 13)(1), an important group of starch-converting enzymes. Catalysis in the ␣ -amylase family proceeds via a covalently linked intermediate (2), which basically divides the reaction in two steps. In the first step the donor substrate (starch or oligosaccharide) is processed, yielding the covalent intermediate (donor reaction). In the second step the acceptor reacts with this intermediate, resulting in product formation (acceptor reaction). Whereas ␣ -amylases usually catalyze a hydrolysis reaction using water as acceptor, CGTases mainly catalyze transglycosylation reactions in which the acceptor is either the non-reducing end glucose of another oligosaccharide (disproportionation) or the non-reducing end glucose of the covalently linked oligosaccharide intermediate, resulting in formation of a cyclodextrin (cyclization). Also the reverse of the cyclization, in which a cyclodextrin is cleaved and transferred to an accepting oligosaccharide (coupling), is catalyzed by CGTase (3). The main determinants that cause the difference in reaction specificity between ␣ -amylases and CGTases are thus likely to be found at the acceptor binding sites.The crystal structures of the Bacillus circulans strain 251 CGTase in complex with an acarbose-derived maltononaose inhibitor (4, 5) and a maltononaose substrate (2) have revealed the nature of the acceptor site. In the maltononaose s...

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Section: Discussionmentioning

confidence: 97%

Hydrophobic Amino Acid Residues in the Acceptor Binding Site Are Main Determinants for Reaction Mechanism and Specificity of Cyclodextrin-glycosyltransferase

Veen¹,

Leemhuis²,

Kralj³

et al. 2001

Journal of Biological Chemistry

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“…It is, therefore, unlikely that, at low pH, protonation of these carboxyl groups in Amy will be inhibitory, and alkaline rather then acidic pH might pose a problem. Recently, Nakamura et al [20] have shown that replacement of the second last, and universally conserved His residue of box 4 by Asn in a CTGase from an alkaliphilic Bacillus reduces the activity of the enzyme at alkaline pH, suggesting that this residue might be involved in catalysis under these conditions [60]. Amy contains glutamine at this position (Gln900, Fig.…”

Section: Discussionmentioning

confidence: 99%

Purification, Properties and Structural Aspects of a Thermoacidophilic α‐Amylase from Alicyclobacillus Acidocaldarius Atcc 27009

Schwermann

Pfau

Liliensiek

et al. 1994

European Journal of Biochemistry

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The a-amylase from the thermoacidophilic eubacterium Alicyclobacillus (Bacillus) acidocaldarius strain ATCC 27009 was studied as an example of an acidophilic protein. The enzyme was purified from the culture fluid. On an SDS/polyacrylamide gel, the protein exhibited an apparent molecular mass of 160 kDa, which is approximately 15% higher than that predicted from the nucleotide sequence. The difference is due to the enzyme being a glycoprotein. Deglycosylation or synthesis of the enzyme in Escherichia coli gave a product with the mass expected for the mature protein.The amylase hydrolyzed starch at random and from the inside, and its main hydrolysis products were maltotriose and maltose. It also formed glucose from starch (by hydrolysing the intermediate product maltotetraose to glucose and maltotriose) and exhibited some pullulanase activity. The pH and temperature optima were pH 3 and 75 "C, respectively, characterizing the enzyme as being thermoacidophilic. Alignment of the sequence of the enzyme with that of its closests neutrophilic relatives and with that of a-1,4 or a-1,6 glycosidic-bond hydrolyzing enzymes of known threedimensional structure showed that the acidophilic a-amylase contains approximately 30% less charged residues than do its closests relatives, that these residues are replaced by neutral polar residues, and that hot spots for these exchanges are likely to be located at the surface of the protein.Literature data show that similar effects are observed in three other acidophilic proteins. It is proposed that these proteins have adapted to the acidic environment by reducing the density of both positive and negative charges at their surface, that this effect circumvents electrostatic repulsion of charged groups at low pH, and thereby contributes to the acidostability of these proteins.Enzymes optimally active under extreme conditions are both of biotechnological importance and of scientific interest (for reviews see [I-31). Much work has been devoted to the mechanism of thermostability. However, its outcome is complex. Theoretical considerations suggest that the exchange of a single amino acid residue in an enzyme can increase its temperature optimum by more than 10°C [3]. Studies on forced molecular evolution have provided support for this notion. In such thermo-drill experiments, a gene encoding a mesophilic enzyme is introduced into a thermophilic organism, and by gradually increasing the growth tem-Correspondence to E. P. Bakker, Abteilung Mikrobiologie,

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“…His-327 is especially important for catalysis over the alkaline pH range. 44) Three-dimensional structures of cyclodextrin glucanotransferases (CGTases) have revealed that four aromatic residues, which are highly conserved among CGTases but not in α-amylases, are located in the active center. Industrial production of cyclodextrins.…”

Section: )mentioning

confidence: 99%

Alkaliphiles

Horikoshi

2004

Proc. Jpn. Acad., Ser. B

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Abstract:The term alkaliphile is used for microorganisms that grow optimally or very well at pH values above 9, but cannot grow or grow only slowly at the near neutral pH value of 6.5. Alkaliphiles include prokaryotes, eukaryotes, and archaea. Alkaliphiles can be isolated from normal environments such as garden soil, although viable counts of alkaliphiles are higher in samples from alkaline environments. The cell surface plays a key role in keeping the intracellular pH value in the range between 7 and 8.5, allowing alkaliphiles to thrive in alkaline environments. Alkaliphiles have made a great impact in industrial applications. Biological detergents contain alkaline enzymes, such as alkaline cellulases and/or alkaline proteases that have been produced from alkaliphiles. Another important application is the industrial production of cyclodextrin with alkaline cyclomaltodextrin glucanotransferase. This enzyme reduced the production cost and paved the way for cyclodextrin use in large quantities in foodstuffs, chemicals and pharmaceuticals. It has also been reported that alkali-treated wood pulp could be biologically bleached by xylanases produced by alkaliphiles.

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Three histidine residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011: effects of the replacement on pH dependence and transition-state stabilization

Cited by 100 publications

References 52 publications

Hydrophobic Amino Acid Residues in the Acceptor Binding Site Are Main Determinants for Reaction Mechanism and Specificity of Cyclodextrin-glycosyltransferase

Hydrophobic Amino Acid Residues in the Acceptor Binding Site Are Main Determinants for Reaction Mechanism and Specificity of Cyclodextrin-glycosyltransferase

Purification, Properties and Structural Aspects of a Thermoacidophilic α‐Amylase from Alicyclobacillus Acidocaldarius Atcc 27009

Alkaliphiles

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