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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
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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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