Search for a dextransucrase minimal motif involved in dextran binding

Abstract

Fourteen truncated forms of Leuconostoc mesenteroides NRRL B512-F dextransucrase, involving N-, C-or N-plus C-terminal domain truncations were tested for their ability to bind dextrans. The shortest fragment (14 kDa molecular weight) that still exhibited a strong interaction with dextran was localized between amino acids N1397 and A1527 of the C-terminal domain (GBD-7) and consists of six YG repeats. With a dissociation constant K d of 2.8 · 10À9 M, this motif shows a very high affinity for isomaltohexaose and longer dextrans, supporting the proposed role of GBD in polymer formation. The potential application of GBD-7 as an affinity tag onto cheap resins like Sephacryl Ò S300HR for rapid purification was evaluated and is discussed.

Investigations on the Determinants Responsible for Low Molar Mass Dextran Formation by DSR-M Dextransucrase

“…Isothermal titration calorimetry, as well as affinity gel electrophoreses, confirmed that DSR-MΔ2 affinity for dextran chains is mainly mediated by domain V, as no binding to dextran could be detected for DSR-MΔV (Figure S7 in the Supporting Information). However, in comparison to the nanomolar affinity estimated for dextran binding of GTF-I and DSR-S domain V fragments, 30,33 DSR-MΔ2 K d values were estimated at 10 mM for I6 and 300 μM for 68.4 kg mol −1 dextran, respectively, which are extremely high but are in agreement with the nonprocessive character of the enzyme.…”

“…reuteri 180 or the alternansucrase ASR from L. mesenteroides NRRL B-1355 synthesize two glucan populations, the first of very high molar mass (around 10 7 g mol –1 ) and the other comprising oligosaccharides or polymers of medium size (10 3 –10 4 g mol –1 ). − Several studies suggest that domain V, located at the N- and C-terminal extremity of the enzymes, plays a role in the polymer size through its capacity to bind glucans. ,, Indeed, domain V fragments are able to fold independently and strongly bind α-glucans with dissociation constants in the nanomolar range. ,− Called accordingly the glucan binding domain (GBD), this domain V contains repeated motifs derived from a common YG motif . The available crystal structures of GH70 enzymes unfortunately mostly concern truncated constructs, in which only a small part of the native domain V is visible. ,, Only very recently, several complexes of the ΔN123-GBD-CD2 branching sucrase with glucose, isomaltose (I2), and isomaltotriose (I3) provided the first structural evidence of sugar binding pockets with a common topology and directly interacting with carbohydrates in domain V .…”

Bacterial α-Glucan and Branching Sucrases from GH70 Family: Discovery, Structure–Function Relationship Studies and Engineering

“…Repeated sequences are found in this domain and their role has been investigated since the 90s [ 92 , 94 , 166 , 169 , 170 ]. Their partial or total truncations influence the glucan binding ability and also modify the size of the synthesized polymer [ 77 , 94 , 119 , 123 , 160 , 171 , 172 , 173 , 174 , 175 , 176 , 177 , 178 ]. In particular, the glucan binding domain was proposed to be a major actor of the processive synthesis of high molar mass dextrans by providing anchoring platform with high affinity for the growing chain [ 119 ].…”

A bio-coupling approach using a dextran-binding domain to immobilize an engineered streptavidin to Sephadex for easy preparation of affinity matrix

“…Since the binding affinity of DBD to dextran is in the nmolar range 6 while the binding strength of agarose-binding domain to agarose is in the µmolar range 4,22 , the retention ability of the bound monomeric or tetrameric DBD to the Sephadex surface is much better than that of the monomeric or tetrameric agarose-binding domain to agarose. This makes DBD an attractive immobilization tool for affinity matrix preparation.…”

“…To extend the fusion protein based bio-coupling approach for affinity matrix preparation, the feasibility to use a dextran-binding domain (DBD) as a matrix immobilization tool was explored. DBD used in this study is a well characterized domain 6 from a monomeric Leuconostoc mesenteroides dextransucrase 7 . It has two key features.…”