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1.

001-es BibID:BIBFORM095731
Első szerző:Kandra Lili (biokémikus)
Cím:Mapping of barley α-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft / Lili Kandra, Maher Abou Hachem, Gyöngyi Gyémánt, Birte Kramhøft, Birte Svensson
Dátum:2006
ISSN:0014-5793
Megjegyzések: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 unfavor able binding energies at the internal subsites -3 and -5 and at subsites -8 and +3/+4 defining these subsites as binding barriers. Barley a-amylase 1 mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 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-amylas.
Tárgyszavak:Természettudományok Biológiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
Barley a-amylase
Subsite mutants
2-Chloro-4-nitrophenyl b-DD-maltooligosaccharides
Bond cleavage frequencies
Subsite maps
Megjelenés:Febs Letters. - 580 : 21 (2006), p. 5049-5053. -
További szerzők:Abou Hachem, Maher Gyémánt Gyöngyi (1960-) (vegyész) Kramhøft, Birte Svensson, Birte
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DOI
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2.

001-es BibID:BIBFORM010529
Első szerző:Nielsen, Morten M.
Cím:Two Secondary Carbohydrate Binding Sites on the Surface of Barley alpha-Amylase 1 Have Distinct Functions and Display Synergy in Hydrolysis of Starch Granules / Morten M. Nielsen, Sophie Bozonnet, Eun-Seong Seo, János A. Mótyán, Joakim M. Andersen, Adiphol Dilokpimol, Maher Abou Hachem, Gyöngyi Gyémánt, Henrik Næsted, Lili Kandra, Bent W. Sigurskjold, Birte Svensson
Dátum:2009
ISSN:0006-2960
Megjegyzések:Some polysaccharide processing enzymes possess secondary carbohydrate binding sites situated on the surface far from the active site. In barley alpha-amylase 1 (AMY1), two such sites, SBS1 and SBS2, are found on the catalytic (beta/alpha)8-barrel and the noncatalytic C-terminal domain, respectively. Site-directed mutagenesis of Trp(278) and Trp(279), stacking onto adjacent ligand glucosyl residues at SBS1, and of Tyr(380) and His(395), making numerous ligand contacts at SBS2, suggested that SBS1 and SBS2 act synergistically in degradation of starch granules. While SBS1 makes the major contribution to binding and hydrolysis of starch granules, SBS2 exhibits a higher affinity for the starch mimic beta-cyclodextrin. Compared to that of wild-type AMY1, the K-d of starch granule binding by the SBS1 W278A, W279A, and W278A/W279A mutants thus increased 15-35 times; furthermore, the k(cat)/K-m of W278A/W279A was 2%, whereas both affinity and activity for Y380A at SBS2 were 10% of the wild-type values. Dual site double and triple SBS1/SBS2 substitutions eliminated binding to starch granules, and the k(cat)/K-m of W278A/W279A/Y380A AMY1 was only 0.4% of the wild-type value. Surface plasmon resonance analysis of mutants showed that beta-cyclodextrin binds to SBS2 and SBS1 with K-d,K-1 and K-d,K-2 values of 0.07 and 1.40 mM, respectively. A model that accounts for the observed synergy in starch hydrolysis., where SBS1 and SBS2 bind ordered and free alpha-glucan chains, respectively, thus targeting the enzyme to single alpha-glucan chains accessible for hydrolysis, is proposed. SBS1 and SBS2 also influence the kinetics of hydrolysis for amylose and maltooligosaccharides, the degree of multiple attack on amylose, and subsite binding energies.
Tárgyszavak:Természettudományok Kémiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Biochemistry. - 48 : 32 (2009), p. 7686-7697. -
További szerzők:Bozonnet, Sophie Seo, Eun-Seong Mótyán János András (1981-) (biokémikus, molekuláris biológus) Andersen, Joakim M. Dilokpimol, Adiphol Abou Hachem, Maher Gyémánt Gyöngyi (1960-) (vegyész) Naested, Henrik Kandra Lili (1943-) (biokémikus) Sigurskjold, Bent W. Svensson, Birte
Internet cím:DOI
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3.

001-es BibID:BIBFORM001054
Első szerző:Nielsen, Morten M.
Cím:Roles of multiple surface sites, long substrate binding clefts, and carbohydrate binding modules in the action of amylolytic enzymes on polysaccharide substrates / M. M. Nielsen, E. S. Seo, A. Dilokpimol, J. Andersen, M. Abou Hachem, H. Naested, M. Willemoës, S. Bozonnet, L. Kandra, G. Gyémánt, R. Haser, N. Aghajari, B. Svensson
Dátum:2008
Megjegyzések:Germinating barley seeds contain multiple forms of alpha-amylases, which are subject to both differential gene expression and differential degradation as part of the repertoire of starch-degrading enzymes. The alpha-amylases are endo-acting and possess a long substrate binding cleft with a characteristic subsite binding energy profile around the catalytic site. Furthermore, several amylolytic enzymes that facilitate attack on the natural substrate, i.e. the endosperm starch granules, have secondary sugar binding sites either situated on the surface of the protein domain or structural unit that contains the catalytic site or belonging to a separate starch binding domain. The role of surface sites in the function of barley a-amylase 1 has been investigated by using mutational analysis in conjunction with carbohydrate binding analyses and crystallography. The ability to bind starch depends on the surface sites and varies for starch granules of different genotypes and botanical origin. The surface sites moreover are candidates for being involved in degradation of polysaccharides by a multiple attack mechanism. Future studies of the molecular nature of the multivalent enzyme-substrate interactions will address surface sites in both barley alpha-amylase 1 and in the related isozyme 2.
Tárgyszavak:Természettudományok Biológiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
amiláz
alhelyek
poliszacharid szubsztrát
felületi plazma rezonancia
Megjelenés:Biocatalysis and Biotransformation. - 26 : 1-2 (2008), p. 59-67. -
További szerzők:Seo, Eun-Seong Dilokpimol, Adiphol Andersen, J. Abou Hachem, Maher Naested, Henrik Willemoës, M. Bozonnet, Sophie Kandra Lili (1943-) (biokémikus) Gyémánt Gyöngyi (1960-) (vegyész) Haser, R. Aghajari, Nushin Svensson, Birte
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4.

001-es BibID:BIBFORM016486
Első szerző:Seo, Eun-Seong
Cím:New Insight into Structure/Function Relationships in Plant alpha-Amylase Family GH13 Members / Eun-Seong Seo, Joakim M. Andersen, Morten M. Nielsen, Malene B. Vester-Christensen, Camilla Christiansen, Johanne M. Jensen, János A. Mótyán, Mikkel A. Glaring, Andreas Blennow, Lili Kandra, Gyöngyi Gyémánt, Štefan Janeček, Richard Haser, Nushin Aghajari, Maher Abou Hachem, Birte Svensson
Dátum:2010
ISSN:1344-7882
Megjegyzések:Two carbohydrate binding surface sites (SBSs) on barley alpha-amylase 1 (AMY1) of glycoside hydrolase family 13 (GH13) displayed synergy in interactions with starch granules, thus being pivotal for hydrolysis of supramolecular substrates. Mutational analysis showed that SBS1 is more critical for the conversion of starch granules, while SBS2 has higher affinity than SBS1 for beta-cyclodextrin (beta-CD). Noticeably, the binding preference of beta-CD to SBS2 differed distinctly from that of maltooligosaccharides to the catalytic nucleophile mutant D180A AMY1. Binding energy maps at subsites -8 through +4 of the active site indicated remarkably elevated affinity due to the Y380A mutation at SBS2. The high-yield AMY2 expression variant A42P, made it possible to show that Tyr378-corresponding to Tyr380 in AMY1-has a role in interactions with starch granules, but not in beta-CD binding. Besides SBSs, dedicated starch binding domains (SBDs) mediate binding to starch granules. SBDs are currently categorised into 9 carbohydrate binding module (CBM) families. A novel CBM20 subfamily encountered in regulatory enzymes possesses characteristically low affinity for beta-CD. Although alpha-amylase is essential for starch mobilisation in germinating barley seeds, efficient degradation requires the concerted action of alpha-amylase, limit dextrinase (LD) and possibly alpha-glucosidase. Limit dextrinase (LD) is encoded by a single gene and represents the sole debranching activity during germination. Recent expression of functional LD in Pichia pastoris makes biochemical and biophysical characterisation of this GH13 enzyme possible. An endogenous limit dextrinase inhibitor was cloned and produced recombinantly and demonstrated to have sub-nanomolar affinity for LD.
Tárgyszavak:Természettudományok Kémiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
surface binding sites, amylose, starch granules, starch binding domain, limit dextrinase
Megjelenés:Journal of applied glycoscience. - 57 : 2 (2010), p. 157-162. -
További szerzők:Andersen, Joakim M. Nielsen, Morten M. Vester-Christensen, Malene B. Christiansen, Camilla Jensen, Johanne M. Mótyán János András (1981-) (biokémikus, molekuláris biológus) Glaring, Mikkel A. Blennow, Andreas Kandra Lili (1943-) (biokémikus) Gyémánt Gyöngyi (1960-) (vegyész) Janeček, Štefan Haser, Richard Aghajari, Nushin Abou Hachem, Maher Svensson, Birte
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