Structure of a<i>Bacillus halmapalus</i>family 13 α-amylase, BHA, in complex with an acarbose-derived nonasaccharide at 2.1 Å resolution

Davies, G.J.; Brzozowski, A.M.; Dauter, Zbigniew; Rasmussen,; Borchert, Torben V.; Wilson, K.S.

doi:10.1107/s0907444904027118

Cited by 28 publications

(31 citation statements)

References 20 publications

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“…Boel et al, 1990;Nielsen et al, 2003), was not observed for AmyD. In the 3D structure of bacterial GH13_5 family enzymes, a triad of metal ions (Ca 2+ -Na + -Ca 2+ ) has been observed between domain A and domain B, and an additional Ca 2+ ion has been located between domain A and domain C (Davies et al, 2005;Machius et al, 1998;Brzozowski et al, 2000). The amino acids interacting with these Ca 2+ ions are only partly conserved among the fungal GH13_5 family enzymes; the presence of bound Ca 2+ ions in these enzymes therefore appears less likely.…”

Section: Discussionmentioning

confidence: 99%

Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal α-amylase enzymes

et al. 2007

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Currently known fungal a-amylases are well-characterized extracellular enzymes that are classified into glycoside hydrolase subfamily GH13_1. This study describes the identification, and phylogenetic and biochemical analysis of novel intracellular fungal a-amylases. The phylogenetic analysis shows that they cluster in the recently identified subfamily GH13_5 and display very low similarity to fungal a-amylases of family GH13_1. Homologues of these intracellular enzymes are present in the genome sequences of all filamentous fungi studied, including ascomycetes and basidiomycetes. One of the enzymes belonging to this new group, Amy1p from Histoplasma capsulatum, has recently been functionally linked to the formation of cell wall a-glucan. To study the biochemical characteristics of this novel cluster of a-amylases, we overexpressed and purified a homologue from Aspergillus niger, AmyD, and studied its activity product profile with starch and related substrates. AmyD has a relatively low hydrolysing activity on starch (2.2 U mg "1 ), producing mainly maltotriose. A possible function of these enzymes in relation to cell wall a-glucan synthesis is discussed. INTRODUCTIONa-Amylases are widely occurring enzymes which hydrolyse the a-(1,4)-glycosidic bonds in starch and glycogen, producing short maltooligosaccharides and maltose. Based on sequence similarity, most a-amylases (EC 3.2.1.1) are classified in glycoside hydrolase (GH) family 13, although some a-amylases originating from extremophilic organisms belong to family GH57 (Henrissat, 1991;Henrissat & Bairoch, 1996) (see also the CAZy website, http://www.cazy.org). Based on a phylogenetic analysis of 1691 different members of the GH13 family, the family has recently been divided into 35 subfamilies, all acting on aglycosidic bonds (Stam et al., 2006). Several of these subfamilies display a-amylase specificity, but many other enzyme reaction specificities are also represented. The tertiary structure of these enzymes is characterized by a (b/a) 8 barrel containing four highly conserved amino acid regions that form the active site (MacGregor et al., 2001), but the overall sequence similarity can be as low as 10 % and only a catalytic triad of amino acids is conserved invariantly (Machovic & Janecek, 2003). The shared (b/a) 8 barrel structure and catalytic mechanism within GH13 enzymes are believed to represent a common evolutionary origin (Kuriki & Imanaka, 1999;Janecek, 1997). The presence of the four conserved regions and a common secondary and tertiary structure allow construction of alignments and phylogenetic studies within the family. The phylogeny of a-amylases is generally in agreement with their origin, e.g. all fungal a-amylases are more related to each other than to the a-amylases originating from plants or animals. a-Amylases from bacteria, however, are scattered over several clusters, which group with animal, plant or fungal a-amylases, or form a separate branch (Janecek, 1994).Several a-amylases from yeasts and fungi have been studied previously (see e.g....

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Section: Discussionmentioning

confidence: 99%

Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal α-amylase enzymes

et al. 2007

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“…Finally, superimposition of barley, B. amyloliquefaciens, B. licheniformis, and B. halmapalus a-amylases, and Bacillus sp. 707 maltohexaose-producing amylase showed a shared S-shaped P 9 subsite cleft [3,5,27,28] with an internal barrier manifested by lack of enzyme-sugar hydrogen bonds at subsite À3. Remarkably, at subsite À6 Trp140 in maltohexaose-producing amylase, a conserved tryptophan in Bacillus a-amylases, and Tyr105 in AMY1 superimpose perfectly with rmsd of back bone atoms <0.1 Å (not shown).…”

Section: Comparison With Other A-amylasesmentioning

confidence: 99%

Mapping of barley α‐amylases and outer subsite mutants reveals dynamic high‐affinity subsites and barriers in the long substrate binding cleft

et al. 2006

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Subsite affinity maps of long substrate binding clefts in barley a-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3-12, revealed unfavorable binding energies at the internal subsites À3 and À5 and at subsites À8 and +3/+4 defining these subsites as binding barriers. Barley a-amylase 1 mutants Y105A and T212Y at subsite À6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (À2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in a-amylases.

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“…Structural studies of ␣-amylases in complex with substrate analogues have received much attention in the past decade (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). The pseudotetrasaccharide inhibitor acarbose was used for the vast majority of these studies.…”

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Oligosaccharide Binding to Barley α-Amylase 1

Robert

Haser

Mori

et al. 2005

Journal of Biological Chemistry

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Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley ␣-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley ␣-amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely ؊7 through ؉2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley ␣-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1 D180A ) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as "molecular tweezers."

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Structure of aBacillus halmapalusfamily 13 α-amylase, BHA, in complex with an acarbose-derived nonasaccharide at 2.1 Å resolution

Cited by 28 publications

References 20 publications

Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal α-amylase enzymes

Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal α-amylase enzymes

Mapping of barley α‐amylases and outer subsite mutants reveals dynamic high‐affinity subsites and barriers in the long substrate binding cleft

Oligosaccharide Binding to Barley α-Amylase 1

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