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Birte Svensson - One of the best experts on this subject based on the ideXlab platform.
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Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley Limit Dextrinase
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2020Co-Authors: Susan Andersen, Birte Svensson, Marie Sofie MøllerAbstract:Abstract Barley Limit Dextrinase (HvLD) of glycoside hydrolase family 13 is the sole enzyme hydrolysing α-1,6-glucosidic linkages from starch in the germinating seed. Surprisingly, HvLD shows 150- and 7-fold higher activity towards pullulan and β-Limit dextrin, respectively, than amylopectin. This is investigated by mutational analysis of residues in the N-terminal CBM-21-like domain (Ser14Arg, His108Arg, Ser14Arg/His108Arg) and at the outer subsites +2 (Phe553Gly) and +3 (Phe620Ala, Asp621Ala, Phe620Ala/Asp621Ala) of the active site. The Ser14 and His108 mutants mimic natural LD variants from sorghum and rice with elevated enzymatic activity. Although situated about 40 A from the active site, the single mutants had 15–40% catalytic efficiency compared to wild type for the three polysaccharides and the double mutant retained 27% activity for β-Limit dextrin and 64% for pullulan and amylopectin. These three mutants hydrolysed 4,6-O-benzylidene-4-nitrophenyl-63-α- d -maltotriosyl-maltotriose (BPNPG3G3) with 51–109% of wild-type activity. The results highlight that the N-terminal CBM21-like domain plays a role in activity. Phe553 and the highly conserved Trp512 sandwich a substrate main chain glucosyl residue at subsite +2 of the active site, while substrate contacts of Phe620 and Asp621 at subsite +3 are less prominent. Phe553Gly showed 47% and 25% activity on pullulan and BPNPG3G3, respectively having a main role at subsite +2. By contrast at subsite +3, Asp621Ala increased activity on pullulan by 2.4-fold, while Phe620Ala/Asp621Ala retained only 7% activity on pullulan albeit showed 25% activity towards BPNPG3G3. This outcome supports that the outer substrate binding area harbours preference determinants for the branched substrates amylopectin and β-Limit dextrin.
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Structural biology of starch-degrading enzymes and their regulation
Current Opinion in Structural Biology, 2016Co-Authors: Marie Sofie Møller, Birte SvenssonAbstract:Starch is a major energy source for all domains of life. Recent advances in structures of starch-degrading enzymes encompass the substrate complex of starch debranching enzyme, the function of surface binding sites in plant isoamylase, details on individual steps in the mechanism of plant disproportionating enzyme and a self-stabilised conformation of amylose accommodated in the active site of plant α-glucosidase. Important inhibitor complexes include a flavonol glycoside, montbretin A, binding at the active site of human pancreatic α-amylase and barley Limit Dextrinase inhibitor binding to the debranching enzyme, Limit Dextrinase using a new binding mode for cereal protein inhibitors.
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Crystal Structure of Barley Limit Dextrinase-Limit Dextrinase Inhibitor (LD-LDI) Complex Reveals Insights into Mechanism and Diversity of Cereal Type Inhibitors
Journal of Biological Chemistry, 2015Co-Authors: Marie Sofie Møller, Johanne Mørch Jensen, Anette Henriksen, Malene Bech Vester-christensen, Maher Abou Hachem, Birte SvenssonAbstract:Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley Limit Dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, and its endogenous inhibitor (LDI) was solved at 2.7 A. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs.
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Oligosaccharide and substrate binding in the starch debranching enzyme barley Limit Dextrinase
Journal of Molecular Biology, 2015Co-Authors: Marie Sofie Møller, Marie Bøjstrup, Ole Hindsgaul, Birte Svensson, Michael Skovbo Windahl, Maher Abou Hachem, Lyann Sim, Monica M. Palcic, Anette HenriksenAbstract:Complete hydrolytic degradation of starch requires hydrolysis of both the α-1,4- and α-1,6-glucosidic bonds in amylopectin. Limit Dextrinase (LD) is the only endogenous barley enzyme capable of hydrolyzing the α-1,6-glucosidic bond during seed germination, and impaired LD activity inevitably reduces the maltose and glucose yields from starch degradation. Crystal structures of barley LD and active-site mutants with natural substrates, products and substrate analogues were sought to better understand the facets of LD–substrate interactions that confine high activity of LD to branched maltooligosaccharides. For the first time, an intact α-1,6-glucosidically linked substrate spanning the active site of a LD or pullulanase has been trapped and characterized by crystallography. The crystal structure reveals both the branch and main-chain binding sites and is used to suggest a mechanism for nucleophilicity enhancement in the active site. The substrate, product and analogue complexes were further used to outline substrate binding subsites and substrate binding restraints and to suggest a mechanism for avoidance of dual α-1,6- and α-1,4-hydrolytic activity likely to be a biological necessity during starch synthesis.
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Structure of the starch-debranching enzyme barley Limit Dextrinase reveals homology of the N-terminal domain to CBM21.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2012Co-Authors: Marie Sofie Møller, Birte Svensson, Maher Abou Hachem, Anette HenriksenAbstract:Barley Limit Dextrinase (HvLD) is a debranching enzyme from glycoside hydrolase family 13 subfamily 13 (GH13_13) that hydrolyses α-1,6-glucosidic linkages in Limit dextrins derived from amylopectin. The structure of HvLD was solved and refined to 1.9 A resolution. The structure has a glycerol molecule in the active site and is virtually identical to the structures of HvLD in complex with the competitive inhibitors α-cyclodextrin and β-cyclodextrin solved to 2.5 and 2.1 A resolution, respectively. However, three loops in the N-terminal domain that are shown here to resemble carbohydrate-binding module family 21 were traceable and were included in the present HvLD structure but were too flexible to be traced and included in the structures of the two HvLD–inhibitor complexes.
Peter W Gous - One of the best experts on this subject based on the ideXlab platform.
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review amylopectin synthesis and hydrolysis understanding isoamylase and Limit Dextrinase and their impact on starch structure on barley hordeum vulgare quality
Trends in Food Science and Technology, 2017Co-Authors: Peter W GousAbstract:Abstract Background Starch contributes to barley grain and malt quality which in turn contributes to beer quality and flavour; through fermentable sugar profiles, rates of fermentation and Mallard reactions. Both amylopectin and amylose are enzymatically degraded to release maltose, maltotriose and higher order sugars. Scope and approach Amylopectin is highly branched [α-(1 → 6) glycoside bond branch points] with numerous short branches while amylose is a long chained polymer with a few side branches. During grain development, the final level of branching is controlled by two enzymes namely; isoamylase and Limit Dextrinase (LD). Mutations in either of these genes can also result in changes to structure, content, and granule formation and size. During the malting free LD will to cleave the α-(1 → 6) bonds but during mashing processes, bound LD is release, resulting in chains of various length available for other starch degrading enzymes to hydrolyse. Findings and conclusions While there is a good understanding of most of the individual aspects in amylopectin formation, structure and degradation; the story remains incomplete, as most of this understanding has been gained from experiments with only a Limited number of barley varieties, Limitations in the technology for structural measurement, and since no data is available to link structure to fermentable sugar profiles.
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Review: Amylopectin synthesis and hydrolysis – Understanding isoamylase and Limit Dextrinase and their impact on starch structure on barley (Hordeum vulgare) quality
Trends in Food Science & Technology, 2017Co-Authors: Peter W Gous, Glen P. FoxAbstract:Abstract Background Starch contributes to barley grain and malt quality which in turn contributes to beer quality and flavour; through fermentable sugar profiles, rates of fermentation and Mallard reactions. Both amylopectin and amylose are enzymatically degraded to release maltose, maltotriose and higher order sugars. Scope and approach Amylopectin is highly branched [α-(1 → 6) glycoside bond branch points] with numerous short branches while amylose is a long chained polymer with a few side branches. During grain development, the final level of branching is controlled by two enzymes namely; isoamylase and Limit Dextrinase (LD). Mutations in either of these genes can also result in changes to structure, content, and granule formation and size. During the malting free LD will to cleave the α-(1 → 6) bonds but during mashing processes, bound LD is release, resulting in chains of various length available for other starch degrading enzymes to hydrolyse. Findings and conclusions While there is a good understanding of most of the individual aspects in amylopectin formation, structure and degradation; the story remains incomplete, as most of this understanding has been gained from experiments with only a Limited number of barley varieties, Limitations in the technology for structural measurement, and since no data is available to link structure to fermentable sugar profiles.
Azar Shahpiri - One of the best experts on this subject based on the ideXlab platform.
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ethephon advances the release time of Limit Dextrinase from gibberellic acid treated aleurone layer
Biocatalysis and agricultural biotechnology, 2017Co-Authors: Samaneh Siapush, Azar ShahpiriAbstract:Abstract The aleurone layer plays a key role in germination by responding to hormone signals from embryo and producing hydrolases. The barley aleurone layer can be separated from the other seed tissues and maintained in culture, allowing the study of the effect of added signaling molecules in an isolated system. In this work, the effect of ethephon on the protein release from gibberellic acid (GA)-treated aleurone layer was presented for two important enzymes in starch degradation: α-amylase and Limit Dextrinase (LD). The results showed that the release of proteins from aleurone layer to culture supernatant was remarkably increased when GA- treated aleurone layer was exposed to 1000–10000 ppm ethephon. Ethephon enhanced the release of α-amylase and advanced the release time of LD from GA-treated aleurone layer to culture supernatant. The results supports that the release of LD is dependent to cell wall degradation which can be induced by the effect of ethephon.
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spatio temporal appearance of α amylase and Limit Dextrinase in barley aleurone layer in response to gibberellic acid abscisic acid and salicylic acid
Journal of the Science of Food and Agriculture, 2015Co-Authors: Azar Shahpiri, Nasim Talaei, Christine FinnieAbstract:BACKGROUND Cereal seed germination involves mobilization of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellin produced by the embryo the aleurone layer synthesizes hydrolases that are secreted to the endosperm for degradation of storage products. In this study analysis of intracellular protein accumulation and release from barley aleurone layers is presented for the important enzymes in starch degradation: α-amylase and Limit Dextrinase (LD). RESULTS Proteins were visualized by immunoblotting in aleurone layers and culture supernatants from dissected aleurone layers incubated up to 72 h with either gibberellic acid (GA), abscisic acid (ABA) or salicylic acid (SA). The results show that α-amylase is secreted from aleurone layer treated with GA soon after synthesis but the release of LD to culture supernatants was significantly delayed and coincided with a general loss of proteins from aleurone layers. CONCLUSIONS Release of LD was found to differ from that of amylase and was suggested to depend on programmed cell death (PCD). Despite detection of intracellular amylase in untreated aleurone layers or aleurone layers treated with ABA or SA, α-amylase was not released from these samples. Nevertheless, the release of α-amylase was observed from aleurone layers treated with GA+ABA or GA+SA. © 2014 Society of Chemical Industry
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Spatio‐temporal appearance of α‐amylase and Limit Dextrinase in barley aleurone layer in response to gibberellic acid, abscisic acid and salicylic acid
Journal of the Science of Food and Agriculture, 2014Co-Authors: Azar Shahpiri, Nasim Talaei, Christine FinnieAbstract:BACKGROUND Cereal seed germination involves mobilization of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellin produced by the embryo the aleurone layer synthesizes hydrolases that are secreted to the endosperm for degradation of storage products. In this study analysis of intracellular protein accumulation and release from barley aleurone layers is presented for the important enzymes in starch degradation: α-amylase and Limit Dextrinase (LD). RESULTS Proteins were visualized by immunoblotting in aleurone layers and culture supernatants from dissected aleurone layers incubated up to 72 h with either gibberellic acid (GA), abscisic acid (ABA) or salicylic acid (SA). The results show that α-amylase is secreted from aleurone layer treated with GA soon after synthesis but the release of LD to culture supernatants was significantly delayed and coincided with a general loss of proteins from aleurone layers. CONCLUSIONS Release of LD was found to differ from that of amylase and was suggested to depend on programmed cell death (PCD). Despite detection of intracellular amylase in untreated aleurone layers or aleurone layers treated with ABA or SA, α-amylase was not released from these samples. Nevertheless, the release of α-amylase was observed from aleurone layers treated with GA+ABA or GA+SA. © 2014 Society of Chemical Industry
Guoping Zhang - One of the best experts on this subject based on the ideXlab platform.
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The expression of the gene associated with β-glucan content, β-amylase and Limit Dextrinase synthesis as affected by post-heading heat stress in barley
2019Co-Authors: Huifang Zhao, Guoping ZhangAbstract:Abstract Background: Malt barley shows a dramatic deterioration of malt quality when exposed to heat or high temperature stress during grain-filling stage (post heading), and global change results in the more frequent occurrence of high temperature, posing a severe threat to high-quality malt barley production. In a previous study, we found heat stress during grain-filling stage caused the significant reduction of kernel weight, and the significant increase of protein and β-glucan content, and β-amylase and Limit Dextrinase (LD) activities, and the effect varied with barley genotypes and the time of heat stress exposure.Results: In this study, we determined the relative expressions of HvCslF6 and HvCslF9 for β-glucan, HvBmy1 for β-amylase and LD gene for Limit Dextrinase of two barley cultivars（ZU9 and Hua30）under the two heat stress (HS) treatments (32/26℃, day/night), initiated from the 7th day (early grain-filling stage) and the14th day (middle grain-filling stage) after heading. In comparison with normal temperature (24/18℃, day/night), HS treatments significantly up-regulated the relative expression of all four genes, and Hua30 being larger than ZU9. The change pattern of each examined gene for the two barley genotypes under heat stress treatments is completely consistent with that of corresponding malt quality trait as affected by heat stress.Conclusion: The results indicate that the enhancement of β-glucan content, and β-amylase and Limit Dextrinase activities under high temperature during grain filling stage is at least in part attributed to increased expression of the relevant genes.
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The relationship of Limit Dextrinase, Limit Dextrinase inhibitor and malt quality parameters in barley and their genetic analysis
Journal of Cereal Science, 2016Co-Authors: Yuqing Huang, Shengguan Cai, Guoping ZhangAbstract:Abstract Limit Dextrinase (LD) and Limit Dextrinase inhibitor (LDI) are the two important traits affecting malt quality. The genetic variation and controlling of LD activity and LDI content in barley grains and malt are not well understood. In this study, we measured LD activity and LDI content in both grains and malt of 68 cultivated barley genotypes. The results show that there is a wide difference among barley genotypes in both LD activity and LDI content. LD in malt is not correlated with LD in grains, but negatively correlated with LDI in malt. LD in malt is positively correlated with diastatic power (DP), Kolbach index (KI) and soluble nitrogen content (SN), and negatively correlated with viscosity (VC). LDI in malt is positively correlated with DP and total nitrogen (TN), and negatively correlated with KI. Association analysis identifies 5 QTLs associated with LD and 3 QTLs associated with LDI in malt. Three major QTLs controlling LD in malt account for 35.7%, 35.7% and 28.4% of phenotypic variation, respectively. A total of 17 QTLs associated with malt quality are identified. The current results address the importance of both LD and LDI in affecting malt quality and the identified QTLs could be useful in barley breeding.
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Genetic diversity and QTL mapping of thermostability of Limit Dextrinase in barley.
Journal of Agricultural and Food Chemistry, 2015Co-Authors: Xiaolei Wang, Guoping Zhang, Shengguan Cai, Zhong-hua Chen, Meixue Zhou, Xuelei Zhang, Fei DaiAbstract:Limit Dextrinase (LD) is an essential amylolytic enzyme for the complete degradation of starch, and it is closely associated with malt quality. A survey of 51 cultivated barley and 40 Tibetan wild barley genotypes showed a wide genetic diversity of LD activity and LD thermostability. Compared with cultivated barley, Tibetan wild barley showed lower LD activity and higher LD thermostability. A doubled haploid population composed of 496 DArT and 28 microsatellite markers was used for mapping Quantitative Trait Loci (QTLs). Parental line Yerong showed low LD activity and high LD thermostability, but Franklin exhibited high LD activity and low LD thermostability. Three QTLs associated with thermostable LD were identified. The major QTL is close to the LD gene on chromosome 7H. The two minor QTLs colocalized with previously reported QTLs determining malt-extract and diastatic power on chromosomes 1H and 2H, respectively. These QTLs may be useful for a better understanding of the genetic control of LD activity and LD thermostability in barley.
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Genetic architecture of Limit Dextrinase inhibitor (LDI) activity in Tibetan wild barley
BMC Plant Biology, 2014Co-Authors: Yuqing Huang, Shengguan Cai, Yong Han, Fei Dai, Guoping ZhangAbstract:Limit Dextrinase inhibitor (LDI) inhibits starch degradation in barley grains during malting because it binds with Limit Dextrinase (LD). There is a wide genetic variation in LDI synthesis and inactivation during barley grain development and germination. However, the genetic control of LDI activity remains little understood. In this study, association analysis was performed on 162 Tibetan wild accessions by using LDI activity, 835 Diversity Arrays Technology (DArT) markers and single nucleotide polymorphisms (SNPs) of the gene HvLDI encoding LDI. Two DArT markers, bpb-8347, bpb-0068, and 31 SNPs of HvLDI were significantly associated with LDI activity, explaining 10.0%, 6.6% and 13.4% of phenotypic variation, respectively. Bpb-8347 is located on chromosome 6H, near the locus of HvLDI, and bpb-0068 is located on 3H. The current results confirmed the locus of the gene controlling LDI activity and identified a new DArT markers associated with LDI activity. The SNPs associated with LDI activity may provide a new insight into the genetic variation of LDI activity in barley grains.
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Association of HvLDI with Limit Dextrinase activity and malt quality in barley
Biotechnology Letters, 2012Co-Authors: Xiaoli Jin, Shengguan Cai, Zhong-hua Chen, Meixue Zhou, Guoping ZhangAbstract:Limit Dextrinase (LD) is a unique de-branching enzyme involved in starch mobilization of barley grains during malting, and closely related to malt quality. Genotypic variation of LD activity is controlled by genetic factors and also affected by environmental conditions. Correlation analysis between LD activity and four malt quality parameters showed that LD activity was positively correlated with diastatic power, Kolbach index and the quality of malt extract, while negatively correlated with viscosity. The structure-based association analysis demonstrated that HvLDI, a gene encoding Limit Dextrinase inhibitor, was a major determinant of LD activity and malt quality. The single nucleotide polymorphisms associated with LD activity could be used in early generation selection for barley breeding.
Marie Sofie Møller - One of the best experts on this subject based on the ideXlab platform.
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Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley Limit Dextrinase
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2020Co-Authors: Susan Andersen, Birte Svensson, Marie Sofie MøllerAbstract:Abstract Barley Limit Dextrinase (HvLD) of glycoside hydrolase family 13 is the sole enzyme hydrolysing α-1,6-glucosidic linkages from starch in the germinating seed. Surprisingly, HvLD shows 150- and 7-fold higher activity towards pullulan and β-Limit dextrin, respectively, than amylopectin. This is investigated by mutational analysis of residues in the N-terminal CBM-21-like domain (Ser14Arg, His108Arg, Ser14Arg/His108Arg) and at the outer subsites +2 (Phe553Gly) and +3 (Phe620Ala, Asp621Ala, Phe620Ala/Asp621Ala) of the active site. The Ser14 and His108 mutants mimic natural LD variants from sorghum and rice with elevated enzymatic activity. Although situated about 40 A from the active site, the single mutants had 15–40% catalytic efficiency compared to wild type for the three polysaccharides and the double mutant retained 27% activity for β-Limit dextrin and 64% for pullulan and amylopectin. These three mutants hydrolysed 4,6-O-benzylidene-4-nitrophenyl-63-α- d -maltotriosyl-maltotriose (BPNPG3G3) with 51–109% of wild-type activity. The results highlight that the N-terminal CBM21-like domain plays a role in activity. Phe553 and the highly conserved Trp512 sandwich a substrate main chain glucosyl residue at subsite +2 of the active site, while substrate contacts of Phe620 and Asp621 at subsite +3 are less prominent. Phe553Gly showed 47% and 25% activity on pullulan and BPNPG3G3, respectively having a main role at subsite +2. By contrast at subsite +3, Asp621Ala increased activity on pullulan by 2.4-fold, while Phe620Ala/Asp621Ala retained only 7% activity on pullulan albeit showed 25% activity towards BPNPG3G3. This outcome supports that the outer substrate binding area harbours preference determinants for the branched substrates amylopectin and β-Limit dextrin.
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Structural biology of starch-degrading enzymes and their regulation
Current Opinion in Structural Biology, 2016Co-Authors: Marie Sofie Møller, Birte SvenssonAbstract:Starch is a major energy source for all domains of life. Recent advances in structures of starch-degrading enzymes encompass the substrate complex of starch debranching enzyme, the function of surface binding sites in plant isoamylase, details on individual steps in the mechanism of plant disproportionating enzyme and a self-stabilised conformation of amylose accommodated in the active site of plant α-glucosidase. Important inhibitor complexes include a flavonol glycoside, montbretin A, binding at the active site of human pancreatic α-amylase and barley Limit Dextrinase inhibitor binding to the debranching enzyme, Limit Dextrinase using a new binding mode for cereal protein inhibitors.
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Crystal Structure of Barley Limit Dextrinase-Limit Dextrinase Inhibitor (LD-LDI) Complex Reveals Insights into Mechanism and Diversity of Cereal Type Inhibitors
Journal of Biological Chemistry, 2015Co-Authors: Marie Sofie Møller, Johanne Mørch Jensen, Anette Henriksen, Malene Bech Vester-christensen, Maher Abou Hachem, Birte SvenssonAbstract:Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley Limit Dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, and its endogenous inhibitor (LDI) was solved at 2.7 A. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs.
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Oligosaccharide and substrate binding in the starch debranching enzyme barley Limit Dextrinase
Journal of Molecular Biology, 2015Co-Authors: Marie Sofie Møller, Marie Bøjstrup, Ole Hindsgaul, Birte Svensson, Michael Skovbo Windahl, Maher Abou Hachem, Lyann Sim, Monica M. Palcic, Anette HenriksenAbstract:Complete hydrolytic degradation of starch requires hydrolysis of both the α-1,4- and α-1,6-glucosidic bonds in amylopectin. Limit Dextrinase (LD) is the only endogenous barley enzyme capable of hydrolyzing the α-1,6-glucosidic bond during seed germination, and impaired LD activity inevitably reduces the maltose and glucose yields from starch degradation. Crystal structures of barley LD and active-site mutants with natural substrates, products and substrate analogues were sought to better understand the facets of LD–substrate interactions that confine high activity of LD to branched maltooligosaccharides. For the first time, an intact α-1,6-glucosidically linked substrate spanning the active site of a LD or pullulanase has been trapped and characterized by crystallography. The crystal structure reveals both the branch and main-chain binding sites and is used to suggest a mechanism for nucleophilicity enhancement in the active site. The substrate, product and analogue complexes were further used to outline substrate binding subsites and substrate binding restraints and to suggest a mechanism for avoidance of dual α-1,6- and α-1,4-hydrolytic activity likely to be a biological necessity during starch synthesis.
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Structure of the starch-debranching enzyme barley Limit Dextrinase reveals homology of the N-terminal domain to CBM21.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2012Co-Authors: Marie Sofie Møller, Birte Svensson, Maher Abou Hachem, Anette HenriksenAbstract:Barley Limit Dextrinase (HvLD) is a debranching enzyme from glycoside hydrolase family 13 subfamily 13 (GH13_13) that hydrolyses α-1,6-glucosidic linkages in Limit dextrins derived from amylopectin. The structure of HvLD was solved and refined to 1.9 A resolution. The structure has a glycerol molecule in the active site and is virtually identical to the structures of HvLD in complex with the competitive inhibitors α-cyclodextrin and β-cyclodextrin solved to 2.5 and 2.1 A resolution, respectively. However, three loops in the N-terminal domain that are shown here to resemble carbohydrate-binding module family 21 were traceable and were included in the present HvLD structure but were too flexible to be traced and included in the structures of the two HvLD–inhibitor complexes.