Schematic representation of GS from LAB. The deduced amino acid sequence of the L. reuteri 121 glucansucrase was used as the template (97). The four different regions shown are (i) the N-terminal signal sequence; (ii) the N-terminal variable region; (iii) the catalytic core; and (iv) the C-terminal GBD. Alignments (SequenceLogo, http://weblogo.berkeley.edu/) are shown of short regions in GS enzymes (GH70) with conserved amino acid residues for which mutant information is available in the literature. The GS protein sequences used are listed in Table Table1.1. The four conserved regions (I to IV) first identified in members of the α-amylase family (GH13) (191) which can also be found in GS enzymes (family GH70) are indicated (see also Fig. Fig.2).2). The seven residues that are fully conserved in family GH13 are also present and fully conserved in the GS family (GH70), except the histidine residue in region I which is present in virtually all family GH13 enzymes but is replaced by a conserved glutamine (Gln1514) in all GS enzymes (110, 121). The seven conserved residues are indicated with arrows, and black arrows indicate the three catalytic residues. (A) Tyr residues, at positions 169 to 172 in GTFB of S. mutans GS5; mutation of these residues into Ala only changed the adhesiveness of the glucan products (202). (B) Thr344Leu, Glu349Leu, and His355Val of GTF-I, causing drops in enzyme activities of 30-, 4-, and 7-fold, respectively (122). (C) Asp511Asn and Asp513Asn, Asp411 and Asp413, and Asp437 and Asp439 of DSRS from L. mesenteroides NRRL B-512F and GTFB and GTFC from S. mutans GS-5, respectively, resulting in complete loss or strongly decreased activities (28, 119). GTFB Val412Ile and GTFC Val438Ile, resulting in enhanced insoluble glucan synthesis of about 10 to 20%, whereas soluble glucan synthesis by these enzymes was significantly lower than for the wild type (28), and GTFB (Glu422Gln) and GTFC (Glu448Gln), resulting in 40% reduced glucan synthesizing activity (28). (D) Catalytic nucleophile in region II, Asp415Asn, and Asp1024Asn (resulting in complete loss of enzyme activity) of GTFI from S. mutans (38) and GTFA from L. reuteri 121 (99). (E) Acid/base catalyst in region III, Glu453Gln, and Glu1061Gln (resulting in complete loss of enzyme activity) of GTFI (38) and GTFA (99), respectively. (F) Transition state stabilizer in region IV, Asp526Asn and Asp1133Asn (resulting in complete loss of enzyme activity) of GTFI (38) and GTFA (99). Other important residues targeted in regions 2D to 2F are discussed in detail in the text. (G) Gln937His in GTFI of Streptococcus downei, resulting in drastic but not complete loss of activity (121).