Deciphering an Undecided Enzyme: Investigations of the Structural Determinants Involved in the Linkage Specificity of Alternansucrase

Understanding how polymerases catalyze the synthesis of biopolymers is a timely and important issue in generating controlled structures with well-defined properties. With this objective in mind, here we describe the 2.8 Å crystal structure of a truncated version of alternansucrase (ASR) from L. citreum NRRL B-1355. Indeed, ASR is a striking example of α-transglucosylase among GH70 glucansucrases, capable of catalyzing high and low molar mass alternan, an α-glucan comprising alternating α-1,3 and α-1,6 linkages in its linear chain. The 3D structure sheds light on the various features involved in enzyme stability. Moreover, docking studies and biochemical characterizations of 17 single mutants and two double mutants enable the key determinants of α-1,6 or α-1,3 linkage specificity to be located and establish the structural basis of alternance. ASR displays two different acceptor subsites in the prolongation of its subsites −1 and +1. The first one is defined by Trp675, a residue of subsite +2, and orients acceptor binding exclusively toward α-1,6 linkage synthesis. The second binding site comprises Asp772 and Trp543, two residues defining the +2′ and +3′ subsites, respectively, which are critical for α-1,3 linkage formation. It is proposed that the interplay between these two acceptor sites controls alternance. These results add to the toolbox of enzymes for the production of tailor-made polysaccharides with controlled structures.