New Insight into Structure/Function Relationships in Plant .ALPHA.-Amylase Family GH13 Members

Seo, Eun-Seong; Andersen, Joakim Mark; Nielsen, Morten; Vester-Christensen, Malene Bech; Christiansen, C; Jensen, Jørgen Dejgaard; Mótyán, János András; Glaring, Mikkel A.; Blennow, Andreas; Kandra, Lili; Gyémánt, Gyöngyi; Janeček, Štefan; Haser, R.; Aghajari, Nushin; Hachem, Maher Abou; Svensson, Birte

doi:10.5458/jag.57.157

Cited by 3 publications

(2 citation statements)

References 35 publications

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“…There is a second isoform AMY2 of this barley enzyme with high sequence identity to AMY1, including key residues in the two SBSs detected in AMY1. SBS2, however, seems non-functional in AMY2 [ 64 ], likely due the altered residues surrounding the conserved SBS2 binding residues [ 65 ]. Thus further testing would be needed to confirm the presence of an SBS in GH5-1 and GH10-1.…”

Section: Discussionmentioning

confidence: 99%

Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

et al. 2016

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Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data.

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Section: Discussionmentioning

confidence: 99%

Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

et al. 2016

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“…While an SBS (SBS1, Fig. 1A) was first discovered in AMY2 (Gibson & Svensson 1987;Kadziola et al 1998), most characterizations of functional properties of SBS1 and SBS2 were carried out with the AMY1 isozyme for two reasons: (i) the yields of recombinant AMY1 produced by Pichia pastoris are about 60 fold higher than of AMY2 (Juge et al 1996); and (ii) preliminary work indicated that binding to SBS2 is weaker in AMY2 (Seo et al 2008(Seo et al , 2010 in agreement with SBS2 also not being occupied in the crystal structure (Kadziola et al 1998). One attractive idea is that this functional difference is of physiological relevance during grain filling and seed germination.…”

Section: Case Stories Of Functional Roles Of Sbss In Gh13 6 and Gh77mentioning

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Analysis of surface binding sites (SBSs) in carbohydrate active enzymes with focus on glycoside hydrolase families 13 and 77 — a mini-review

et al. 2014

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Surface binding sites (SBSs) interact with carbohydrates outside of the enzyme active site. They are frequently situated on catalytic domains and are distinct from carbohydrate binding modules (CBMs). SBSs are found in a variety of enzymes and often seen in crystal structures. Notably about half of the > 45 enzymes (in 17 GH and two GT families) with an identified SBS are from GH13 and a few from GH77, both belonging to clan GH-H of carbohydrate active enzymes. The many enzymes of GH13 with SBSs provide an opportunity to analyse their distribution within this very large and diverse family. SBS containing enzymes in GH13 are spread among 15 subfamilies (two were not assigned a subfamily). Comparison of these SBSs reveals a complex evolutionary history with evidence of conservation of key residues and/or structural location between some SBSs, while others are found at entirely distinct structural locations, suggesting convergent evolution. An array of investigations of the two SBSs in barley α-amylase demonstrated they play different functional roles in binding and degradation of polysaccharides. MalQ from Escherichia coli is an α-1,4-glucanotransferase of GH77, a family that is known to have at least one member that contains an SBS. Whereas MalQ is a single domain enzyme lacking CBMs, its plant orthologue DPE2 contains two N-terminal CBM20s. Surface plasmon resonance binding studies showed that MalQ and DPE2 have a similar affinity for β-cyclodextrin and that MalQ binds malto-oligosaccharides of >DP4 at a second site in competition with β-cyclodextrin yielding a stoichiometry >1. This suggests that MalQ may have an SBS, though its structural location remains unknown.

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Identification, Molecular Cloning, and Recombinant Gene Expression of an Extracellular A-Amylase fromDactylis GlomerataL. Embryogenic Suspension Cultures

Rakleova

Keightley

Pantchev

et al. 2012

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New Insight into Structure/Function Relationships in Plant .ALPHA.-Amylase Family GH13 Members

Cited by 3 publications

References 35 publications

Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

Analysis of surface binding sites (SBSs) in carbohydrate active enzymes with focus on glycoside hydrolase families 13 and 77 — a mini-review

Identification, Molecular Cloning, and Recombinant Gene Expression of an Extracellular A-Amylase fromDactylis GlomerataL. Embryogenic Suspension Cultures

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