Amylomaltase of<i>Pyrobaculum aerophilum</i>IM2 Produces Thermoreversible Starch Gels

Kaper, Thijs; Talik, Boguslawa; Ettema, Thijs J. G.; Bos, H.T.P.; Maarel, Marc J. E. C. van der; Dijkhuizen, Lubbert

doi:10.1128/aem.71.9.5098-5106.2005

Cited by 89 publications

(81 citation statements)

References 58 publications

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“…Similar substrate specificity has been reported for the amylomaltase from Thermus thermophilus HB8 (15). On the contrary, the descending order of preferred substrate is different from that of the Pyrobaculum aerophilum IM2 amylomaltase, which was G3 Ͼ G4 Ͼ G7 Ͼ G5 Ͼ G6 (16). When the kinetic parameters of the WT and Y172A CgAMs were analyzed, the lower K m of the Y172A enzyme suggests that the enzyme-substrate binding affinity is increased in the mutant, while the reaction rate k cat decreases.…”

Section: Discussionmentioning

confidence: 64%

Altered Large-Ring Cyclodextrin Product Profile Due to a Mutation at Tyr-172 in the Amylomaltase of Corynebacterium glutamicum

Srisimarat

Kaulpiboon

Krusong

et al. 2012

Appl Environ Microbiol

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c Corynebacterium glutamicum amylomaltase (CgAM) catalyzes the formation of large-ring cyclodextrins (LR-CDs) with a degree of polymerization of 19 and higher. The cloned CgAM gene was ligated into the pET-17b vector and used to transform Escherichia coli BL21(DE3). Site-directed mutagenesis of Tyr-172 in CgAM to alanine (Y172A) was performed to determine its role in the control of LR-CD production. Both the recombinant wild-type (WT) and Y172A enzymes were purified to apparent homogeneity and characterized. The Y172A enzyme exhibited lower disproportionation, cyclization, and hydrolysis activities than the WT. The k cat /K m of the disproportionation reaction of the Y172A enzyme was 2.8-fold lower than that of the WT enzyme. The LR-CD product profile from enzyme catalysis depended on the incubation time and the enzyme concentration. Interestingly, the Y172A enzyme showed a product pattern different from that of the WT CgAM at a long incubation time. The principal LR-CD products of the Y172A mutated enzyme were a cycloamylose mixture with a degree of polymerization of 28 or 29 (CD28 or CD29), while the principal LR-CD product of the WT enzyme was CD25 at 0.05 U of amylomaltase. These results suggest that Tyr-172 plays an important role in determining the LR-CD product profile of this novel CgAM.

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Section: Discussionmentioning

confidence: 64%

Altered Large-Ring Cyclodextrin Product Profile Due to a Mutation at Tyr-172 in the Amylomaltase of Corynebacterium glutamicum

Srisimarat

Kaulpiboon

Krusong

et al. 2012

Appl Environ Microbiol

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“…Kaper et al (28,29) determined the K m for maltotriose with amylomaltases from T. thermophilus and P. aerophilum to be 2.2 and 3.7 mM, respectively. We obtained a K m of 3.16 mM for maltotriose with ⌬N130 (Fig.…”

Section: Discussionmentioning

confidence: 99%

Domain Characterization of a 4-α-Glucanotransferase Essential for Maltose Metabolism in Photosynthetic Leaves

Steichen

Petty

Sharkey

2008

Journal of Biological Chemistry

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Maltose metabolism during the conversion of transitory (leaf) starch to sucrose requires a 4-␣-glucanotransferase (EC 2.4.1.25) in the cytosol of leaf cells. This enzyme is called DPE2 because of its similarity to the disproportionating enzyme in plastids (DPE1). DPE1 does not use maltose; it primarily transfers a maltosyl unit from one maltotriose to a second maltotriose to make glucose and maltopentaose. DPE2 is a modular protein consisting of a family 77 glycosyl hydrolase domain, similar to DPE1, but unlike DPE1 the domain is interrupted by an insertion of ϳ150 amino acids as well as an N-terminal extension that consists of two carbohydrate binding modules. Phylogenetic analysis shows that the DPE2-type enzyme is present in a limited but highly diverse group of organisms. Here we show that DPE2 transfers the non-reducing glucosyl unit from maltose to glycogen by a ping-pong mechanism. The forward reaction (consumption of maltose) is specific for the ␤-anomer of maltose, while the reverse reaction (production of maltose) is not stereospecific for the acceptor glucose. Additionally, through deletion mutants we show that the glycosyl hydrolase domain alone provides disproportionating activity with a much higher affinity for short maltodextrins than the complete wild-type enzyme, while absence of the carbohydrate binding modules completely abolishes activity with large complex carbohydrates, reflecting the presumed function of DPE2 in vivo.During photosynthesis, as much as one-half of the carbon fixed during the day is stored as transitory starch inside chloroplasts for remobilization at night. The pathway by which starch in leaves is converted to sucrose has only recently been elucidated (1) and some questions remain. The breakdown of storage starch upon seed germination takes place essentially outside of cells (cellular integrity is lost) while transitory starch must be broken down in intact chloroplasts and cells (2). Transitory starch must have some phosphate attached in order to be remobilized at night (3-6). During the day, carbon is exported from the chloroplast almost exclusively as triose phosphate, but at night maltose is exported (7,8). Starch is acted on by ␤-amylase (9), and the resulting ␤-maltose is exported from chloroplasts through a novel maltose transporter (10). Plants lacking this transporter accumulate maltose in their plastids (11). In the cytosol, maltose metabolism requires a 4-␣-glucanotransferase (EC 2.4.1.25). This enzyme was called the cytosolic D enzyme and, by analogy with the plastidial D enzyme, was believed to have no activity with maltose (12). Arabidopsis thaliana plants lacking this enzyme accumulate up to 100-fold more maltose than wild type and grow significantly more slowly (13,14). Potato plants in which this enzyme was reduced in activity accumulated high levels of maltose in leaves but leaf starch synthesis and starch metabolism in tubers was not affected (15).This enzyme is now called disproportionating enzyme 2 (DPE2) 3 because of its similarity to the disproportionat...

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“…These properties, amylose disproportionation and the synthesis of large cyclic glucans, make these enzymes interesting biocatalysts for the synthesis of valuable fine chemicals and pharmaceutically important materials (12)(13)(14).…”

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confidence: 99%

Three-way Stabilization of the Covalent Intermediate in Amylomaltase, an α-Amylase-like Transglycosylase

Barends

Bultema

Kaper

et al. 2007

Journal of Biological Chemistry

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Amylomaltases are glycosyl hydrolases belonging to glycoside hydrolase family 77 that are capable of the synthesis of large cyclic glucans and the disproportionation of oligosaccharides. Using protein crystallography, we have generated a flip book movie of the amylomaltase catalytic cycle in atomic detail. The structures include a covalent glycosyl enzyme intermediate and a covalent intermediate in complex with an analogue of a cosubstrate and show how the structures of both enzyme and substrate respond to the changes required by the catalytic cycle as it proceeds. Notably, the catalytic nucleophile changes conformation dramatically during the reaction. Also, Gln-256 on the 250s loop is involved in orienting the substrate in the ؉1 site. The absence of a suitable base in the covalent intermediate structure explains the low hydrolysis activity.Amylomaltases are 4-␣-glucanotransferase enzymes (1-4) that are structurally and mechanistically related to ␣-amylases (family 13 of the glycoside hydrolases or GH13) (5-7) despite their classification in a separate glycoside hydrolase family (glycoside hydrolase family 77). However, amylomaltases almost exclusively catalyze transglycosylation reactions, whereas ␣-amylase-like enzymes mostly catalyze hydrolysis. As a result, the amylomaltases perform so-called disproportionation reactions, the net effect of which is that two amylose chains of certain lengths react in such a way that one of the chains (the "donor") becomes shorter and the other one (the "acceptor") longer as shown in Reaction 1.Instead of using two different sugar chains for this reaction, the enzyme can also take the non-reducing end of the donor substrate as the acceptor substrate, which results in the formation of a cyclic glucan product of at least 16 glucose units (8 -11). These properties, amylose disproportionation and the synthesis of large cyclic glucans, make these enzymes interesting biocatalysts for the synthesis of valuable fine chemicals and pharmaceutically important materials (12)(13)(14). Over the years, several incisive studies have contributed to our current understanding of catalysis by ␣-amylase-type enzymes. Importantly, a structure of a covalent reaction intermediate, obtained by Uitdehaag et al. (15), provided a detailed look into the active site, showing among other things how the (mutated) acid/base catalyst is poised to activate a water molecule for attack on the C 1 carbon atom. Since then, two other examples of covalent glycosyl enzyme intermediate structures of ␣-amylase-type enzymes have been published (16,17). These structures provided insight into the hydrolysis mechanism, i.e. in the way in which a covalent intermediate is attacked by a water molecule. Three conserved carboxylic acid residues play a central role in the catalytic mechanism (15, 18 -20). The first carboxylic acid residue acts as an acid/base catalyst that protonates the oxygen atom of the scissile glycosidic bond. Simultaneously, the C 1 carbon atom is attacked by the second carboxylate, the nucleophile, which...

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Amylomaltase ofPyrobaculum aerophilumIM2 Produces Thermoreversible Starch Gels

Cited by 89 publications

References 58 publications

Altered Large-Ring Cyclodextrin Product Profile Due to a Mutation at Tyr-172 in the Amylomaltase of Corynebacterium glutamicum

Altered Large-Ring Cyclodextrin Product Profile Due to a Mutation at Tyr-172 in the Amylomaltase of Corynebacterium glutamicum

Domain Characterization of a 4-α-Glucanotransferase Essential for Maltose Metabolism in Photosynthetic Leaves

Three-way Stabilization of the Covalent Intermediate in Amylomaltase, an α-Amylase-like Transglycosylase

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