Thermodynamics of the Binding of the C-Terminal Repeat Domain ofStreptococcus sobrinusGlucosyltransferase-I to Dextran

Abstract

Glucosyltransferases (GTFs) secreted by mutans streptococci and some other lactic acid bacteria catalyze glucan synthesis from sucrose, and possess a C-terminal glucan-binding domain (GBD) containing homologous, directly repeating units. We prepared a series of C-terminal truncated forms of the GBD of Streptococcus sobrinus GTF-I and studied their binding to dextran by isothermal titration calorimetry. The binding of all truncates was strongly exothermic. Their titration curves were analyzed assuming that the GBD recognizes and binds to a stretch of dextran chain, not to a whole dextran molecule. Both the number of glucose units constituting the dextran stretch (n) and the accompanying enthalpy change (DeltaH degrees ) are proportional to the molecular mass of the GBD truncate, with which the Gibbs energy change calculated by the relation DeltaG degrees = -RT ln K (R, the gas constant; T, the absolute temperature; K, the binding constant of a truncate for a dextran stretch of n glucose units) also increases linearly. For the full-length GBD (508 amino acid residues), n = 33.9, K = 4.88 x 10(7) M-1, and DeltaH degrees = -289 kJ mol-1 at 25 degrees C. These results suggest that identical, independent glucose-binding subsites, each comprising 14 amino acid residues on average, are arranged consecutively from the GBD N-terminus. Thus, the GBD binds tightly to a stretch of dextran chain through the adding up of individually weak subsite/glucose interactions. Furthermore, the entropy change accompanying the GBD/dextran interaction as given by the relation DeltaS degrees = (DeltaG degrees - DeltaH degrees)/T has a very large negative value, probably because of a loss of the conformational freedom of dextran and GBD after binding.

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 .…”

Kinetics of dextran‐independent α‐(1→3)‐glucan synthesis by Streptococcus sobrinus glucosyltransferase I

“…Additional studies are required to elucidate the dextran‐dependent synthesis of glucan. As the affinity of the GBd of GTF‐I for dextran is extraordinarily high ( K m = 4.88 × 10 7 m −1 ) [27], the enzyme can hardly be dissociated from dextran. GTF‐I may elongate the α‐(1→3)‐glucose branches of dextran in a processive‐like manner.…”

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 ].…”