Structure-function relationships of glucansucrase and fructansucrase enzymes from lactic acid bacteria - PubMed (original) (raw)
Review
Structure-function relationships of glucansucrase and fructansucrase enzymes from lactic acid bacteria
Sacha A F T van Hijum et al. Microbiol Mol Biol Rev. 2006 Mar.
Abstract
Lactic acid bacteria (LAB) employ sucrase-type enzymes to convert sucrose into homopolysaccharides consisting of either glucosyl units (glucans) or fructosyl units (fructans). The enzymes involved are labeled glucansucrases (GS) and fructansucrases (FS), respectively. The available molecular, biochemical, and structural information on sucrase genes and enzymes from various LAB and their fructan and alpha-glucan products is reviewed. The GS and FS enzymes are both glycoside hydrolase enzymes that act on the same substrate (sucrose) and catalyze (retaining) transglycosylation reactions that result in polysaccharide formation, but they possess completely different protein structures. GS enzymes (family GH70) are large multidomain proteins that occur exclusively in LAB. Their catalytic domain displays clear secondary-structure similarity with alpha-amylase enzymes (family GH13), with a predicted permuted (beta/alpha)(8) barrel structure for which detailed structural and mechanistic information is available. Emphasis now is on identification of residues and regions important for GS enzyme activity and product specificity (synthesis of alpha-glucans differing in glycosidic linkage type, degree and type of branching, glucan molecular mass, and solubility). FS enzymes (family GH68) occur in both gram-negative and gram-positive bacteria and synthesize beta-fructan polymers with either beta-(2-->6) (inulin) or beta-(2-->1) (levan) glycosidic bonds. Recently, the first high-resolution three-dimensional structures have become available for FS (levansucrase) proteins, revealing a rare five-bladed beta-propeller structure with a deep, negatively charged central pocket. Although these structures have provided detailed mechanistic insights, the structural features in FS enzymes dictating the synthesis of either beta-(2-->6) or beta-(2-->1) linkages, degree and type of branching, and fructan molecular mass remain to be identified.
Figures
FIG. 1.
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,
) 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 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. 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).
FIG. 2.
Topology diagrams of members of α-amylase family GH13 (A) and GS proteins of family GH70 (B). The catalytic domain of α-amylases has a (β/α)8 barrel structure, starting with β-strand 1 and ending with α-helix 8. The B domain is located between β-strand 3 and α-helix 3. GS have a putative circularly permuted (β/α)8 barrel structure (111), which starts with α-helix 3 (α-amylase numbering) and ends with β-strand 3. Between α-helix 8 and β-strand 1, a large stretch of unknown function is located. The locations of the four conserved regions (I to IV) in family GH13 (and family GH70) are indicated with dashed boxes. Amylosucrase has a domain loop (B′ domain; important for polymerizing activity; indicated with a dashed line and circle) consisting of approximately 60 amino acid residues which is located after β-strand 7 (182, 183), immediately after the two catalytically important His392 and Asp393 residues located in conserved region IV (165) (Fig. 1). GS proteins also contain an additional “loop” (about 45 amino acids; indicated with a dashed line) compared to α-amylase enzymes, which is located between β-strand 7 and α-helix 7. Conceivably, this loop is also important for polymerizing activity. The approximate sites of the three catalytic residues (D, E, and D) are indicated. B, B domain; C, C domain; GBD, glucan binding domain; VR, variable region. (Adapted from reference with permission of the publisher. Copyright 2005 American Chemical Society.)
FIG. 3.
Schematic representation of FS proteins from LAB. The L. reuteri 121 inulosucrase (Inu) deduced amino acid sequence was used as the template (AF459437). 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 variable region (which in some cases contain an LPXTG cell wall anchor). Alignments (SequenceLogo,
) are shown of short regions in LAB FS protein amino acid sequences (Table 2) with conserved amino acid residues for which mutant information is available in literature. (A) Asp86 in B. subtilis SacB identified as the nucleophile based on crystal structure data for (inactive) mutant Glu342Ala with a bound sucrose (113) and biochemical characterization of the L. reuteri 121 Inu Asp272Asn mutant (140). (B) Mutant Glu117Gln in Z. mobilis SacB, resulting in a higher transglycosylation activity (220); SGSA, sucrose binding box 1, a region conserved between sucrose-utilizing enzymes (167). (C) Mutant Asp312Ser in S. salivarius ATCC 25975 FS (possibly involved in acceptor recognition and/or stabilizing a beta turn in the protein) (185). (D) “Sucrose binding box 2,” a region conserved between sucrose-utilizing enzymes (167). (E) RDP motif, with mutant Asp397Ser in S. salivarius ATCC 25975 FS resulting in complete loss of sucrose hydrolysis and polymerization activities (185), mutant Asp309Asn in G. diazotrophicus LsdA, with a 75-fold reduced catalytic activity (9), mutant Asp194Asn in Z. mobilis SacB, with a 3,400-fold decrease in catalytic activity (220), and Asp247 in B. subtilis SacB and Asp272 in L. reuteri 121 Inu (stabilizer of the oxocarbenium-like transition state) (113, 140). (F) Mutant Glu211Gln in Z. mobilis SacB, with sucrose hydrolysis reduced to 28%, and highly reduced transfructosylation activity. (G) Mutant Glu278Asp (30-fold lower catalytic activity), mutant Glu278His (virtually inactive enzyme) in Z. mobilis levansucrase SacB (220), and Glu342 in B. subtilis SacB and Glu523 in L. reuteri 121 Inu (acid/base catalyst) (113, 140). (H) Arg331His (higher oligosaccharide formation) in levansucrase from B. subtilis SacB (22).
FIG. 4.
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 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. 3A), the neighboring Trp residue (Trp85 in SacB, Trp134 in LsdA, and Trp271 in Inu) (Fig. 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. 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. 3B). The +1 subsite is constituted of Arg/His (Arg360 in SacB, His419 in LsdA, and Arg541 in Inu) (Fig. 3H), Glu/Gln located two residues upstream of the acid-base catalyst (Glu340 in SacB, Gln399 in LsdA, and Glu521 in Inu) (Fig. 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. 3E).
References
- Aduse-Opoku, J., M. L. Gilpin, and R. R. B. Russell. 1989. Genetic and antigenic comparison of Streptococcus mutans fructosyltransferase and glucan-binding protein. FEMS Microbiol. Lett. 50:279-282. - PubMed
- Albenne, C., L. K. Skov, O. Mirza, M. Gajhede, G. Feller, S. D'Amico, G. Andre, G. Potocki-Veronese, B. A. Van Der Veen, P. F. Monsan, and M. Remaud-Simeon. 2004. Molecular basis of the amylose-like polymer formation catalyzed by Neisseria polysaccharea amylosucrase. J. Biol. Chem. 279:726-734. - PubMed
- Arguello-Morales, M. A., M. Remaud-Simeon, S. Pizzut, P. Sarcabal, and P. F. Monsan. 2000. Sequence analysis of the gene encoding alternansucrase, a sucrose glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355. FEMS Microbiol. Lett. 182:81-85. - PubMed
- Arguello-Morales, M. A., M. Remaud-Simeon, R. M. Willemot, M. R. Vignon, and P. F. Monsan. 2001. Novel oligosaccharides synthesized from sucrose donor and cellobiose acceptor by alternansucrase. Carbohydr. Res. 331:403-411. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Miscellaneous