Close-up view of the active site of mutant Glu342Ala B. subtilis SacB levansucrase with a bound sucrose molecule (structure accession code 1PT2). The model was created using SwissPdb Viewer (62). Hydrogen bonds are shown by dashed lines (based on reference 113). Numbering of amino acid residues is based on B. subtilis SacB, with the numbering of L. reuteri 121 Inu (Table (Table2)2) in parentheses. Based on structural information about the SacB (113) and G. diazotrophicus LsdA (112) three-dimensional structures and alignments of FS amino acid sequences the −1 (underlined) and +1 (italics) subsites of family GH68 enzymes were identified (this work). The −1 subsite is constituted of the catalytic nucleophile (Asp86 in SacB, Asp135 in LsdA, and Asp272 in Inu) (Fig. (Fig.3A),3A), the neighboring Trp residue (Trp85 in SacB, Trp134 in LsdA, and Trp271 in Inu) (Fig. (Fig.3A),3A), two residues located in the RDP motif that is conserved in most members of family GH68 (Arg246, Asp247 in SacB, Arg308, Asp309 in LsdA, and Arg423, Asp424 in Inu) (Fig. (Fig.3E),3E), and a Trp residue bordering the sucrose binding pocket and located very close to the fructose moiety at −1 (Trp163 in SacB, Trp224 in LsdA, and Trp340 in Inu) (Fig. (Fig.3B).3B). The +1 subsite is constituted of Arg/His (Arg360 in SacB, His419 in LsdA, and Arg541 in Inu) (Fig. (Fig.3H),3H), Glu/Gln located two residues upstream of the acid-base catalyst (Glu340 in SacB, Gln399 in LsdA, and Glu521 in Inu) (Fig. (Fig.3G),3G), and Arg from the RDP motif (also involved in formation of the −1 subsite) (Arg246 in SacB, Arg308 in LsdA and Arg423 in Inu) (Fig. (Fig.3E3E).