Wikipedia

Glucansucrase

Glucansucrase(also known asglucosyltransferase) is anenzymein theglycoside hydrolasefamily GH70 used bylactic acidbacteriato splitsucrose;it then utilizes the resultingglucosemoleculesto build long, stickybiofilmchains. Theseextracellularhomopolysaccharides are called α-glucanpolymers.

Glucansucrase inStreptococcus mutans.The domains are color coded. For domains made up of discontiguous segments, each segment was assigned a number. Shown here are segments IV1 (orange), B1 (red), A1 (blue), C (pink), A2 (purple), B2 (yellow), and IV2 (green).

Glucansucrase enzymes can synthesize a variety of glucans with differingsolubilities,rheology,and other properties by altering the type of glycosidic linkage, degree of branching, length, mass, and conformation of the polymers. Glucansucrases are classified according to the glycosidic linkage they catalyze. They can be mutansucrases, dextransucrases, alternansucrases, or reuteransucrases.[1]This versatility has made glucansucrase useful for industrial applications.[2]Glucansucrase's role incariogenesisis a major point of interest. Glucan polymers stick to teeth in the human mouth and causetooth decay.[3]

Structure

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Glucansucrases are large, extracellular proteins with average molecular masses around 160,000daltons.Thereforecrystallographystudies have only been carried out for fragments of the enzymes, not complete structures. However, glucansucrase is very similar toα-amylase,another sugar-cutting enzyme.[2]Glucansucrase thus has many of the same structural features. For example, both enzymes have three domains in their catalytic core and a (β/α)8barrel.[4]

Glucansucrase has five major domains: A, B, C, IV, and V. The domains in glucansucrase, however, have a different arrangement than those in α-amylase. The folding characteristics of α-amylase and glucansucrase are still very similar, but their domains are permuted.[5][6][3] Domains A, B, IV, and V are built from two discontiguous parts of the polypeptide chain, causing the chain to follow a U-shape.[1]From the N- to C-terminus, the polypeptide chain goes in the following order: V, IV, B, A, C, A, B, IV, V (see figure at top right).[4]The C domain is the only one made up of a continuous polypeptide sequence.

Domain A contains the (β/α)8barrel and the catalytic site. In the catalytic site, threeresiduesin particular play important roles for enzymatic activity: anucleophilicaspartate,an acid/baseglutamate,and an additional aspartate to stabilize thetransition state.[4][3]

Domain B makes up a twisted antiparallelβ sheet.Some of the loops in domain B help shape the groove near the catalytic site. Additionally, some amino acids between domains A and B form acalciumbinding site near the nucleophilic aspartate. The Ca2+ion is necessary for enzyme activity.[4][3]

Reaction and Mechanism

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Glucansucrase has two parts to its reaction. First it cleaves aglycosidic bondto split sucrose. Products of the reaction are the constituentmonosaccharidesglucose andfructose.This glucose is added to a growing glucan chain. Glucansucrase uses the energy released from bond cleavage to drive glucan synthesis.[2]Both sucrose breakdown and glucan synthesis occur in the same active site.[3]

The first step is carried out through a transglycosylation mechanism involving a glycosyl-enzymeintermediatein subsite-1. Glutamate is likely the catalytic acid/base, aspartate the nucleophile, and another aspartate the transition state stabilizer.[5][7]These three residues are all highly conserved and mutating them leads to a significant decrease in enzymatic activity.[3]

Active site of glucansucrase inLactobacillus reuteri

The glucansucrase mechanism has historically been controversial in the scientific literature.[8][9][10]The mechanism involves two displacements. The first originates from a glycosidic cleavage of the sucrose substrate between subsites -1 and +1. This releases fructose and forms a sugar-enzyme intermediate when the glucose unit attaches to the nucleophile.

The second displacement is transfer of a glucosylmoietyto an acceptor, such as a growing glucan chain. The debate in the past was over whether the glucosyl group attached to the non-reducing or reducing end of an incoming acceptor. Additional investigations pointed to a non-reducing mechanism with a single active site.[1][2][11][3]

Evolution

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Glucansucrase proteins likely evolved from an amylase enzyme precursor.[3]The two enzymes have similar folding patterns and protein domains. In fact, past attempts to produce drugs targeting glucansucrase have not been successful because the drugs also disrupted amylase, which is necessary to break downstarches.[12][13]This occurred because the active sites of the two enzymes are nearly the same. Glucansucrase likely maintained a highly-conserved active site as it underwent a different evolutionary path.

Health

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Glucansucrase allows the oral bacteriaStreptococcus mutansto metabolize sucrose into lactic acid. This lactic acid lowers thepHaround teeth and dissolvescalcium phosphateintooth enamel,leading to tooth decay.[14]Additionally, the synthesis of glucan aidsS. mutansin adhering to the surface of teeth.[15][16]As the polymers accumulate, they help more acid-producing bacteria stay on teeth. Consequently, glucansucrase is such an attractive drug target to prevent tooth decay. IfS. mutanscan no longer break down sucrose and synthesize glucan, calcium phosphate is not degraded and bacteria cannot adhere as easily to teeth.

Industry

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Bacteria with glucansucrase enzymes are used extensively in industry for a variety of applications. The polymerdextranis one prominent example of a very useful polymer. It is produced at commercial scale for uses inveterinary medicine,separation technology,biotechnology,the food industry for gelling, viscosifying, and emulsifying, in human medicine as aprebiotic,cholesterol-lowering agent orblood plasmaexpander, and more.[4][8][17]

See also

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References

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  1. ^abcIto K, Ito S, Shimamura T, Weyand S, Kawarasaki Y, Misaka T, Abe K, Kobayashi T, Cameron AD, Iwata S (April 2011). "Crystal structure of glucansucrase from the dental caries pathogen Streptococcus mutans".Journal of Molecular Biology.408(2): 177–86.doi:10.1016/j.jmb.2011.02.028.PMID21354427.
  2. ^abcdvan Hijum SA, Kralj S, Ozimek LK, Dijkhuizen L, van Geel-Schutten IG (March 2006)."Structure-function relationships of glucansucrase and fructansucrase enzymes from lactic acid bacteria".Microbiology and Molecular Biology Reviews.70(1): 157–76.doi:10.1128/MMBR.70.1.157-176.2006.PMC1393251.PMID16524921.
  3. ^abcdefghVujicic-Zagar A, Pijning T, Kralj S, López CA, Eeuwema W, Dijkhuizen L, et al. (December 2010)."Crystal structure of a 117 kDa glucansucrase fragment provides insight into evolution and product specificity of GH70 enzymes".Proceedings of the National Academy of Sciences of the United States of America.107(50): 21406–11.Bibcode:2010PNAS..10721406V.doi:10.1073/pnas.1007531107.PMC3003066.PMID21118988.
  4. ^abcdeLeemhuis H, Pijning T, Dobruchowska JM, van Leeuwen SS, Kralj S, Dijkstra BW, Dijkhuizen L (January 2013). "Glucansucrases: three-dimensional structures, reactions, mechanism, α-glucan analysis and their implications in biotechnology and food applications".Journal of Biotechnology.163(2): 250–72.doi:10.1016/j.jbiotec.2012.06.037.PMID22796091.
  5. ^ab"Glucansucrase".PDB101: Molecule of the Month.
  6. ^MacGregor EA, Jespersen HM, Svensson B (January 1996). "A circularly permuted Alpha -amylase-type Alpha /beta-barrel structure in glucan-synthesizing glucosyltransferases".FEBS Letters.378(3): 263–6.Bibcode:1996FEBSL.378..263M.doi:10.1016/0014-5793(95)01428-4.PMID8557114.
  7. ^Tsumori H, Minami T, Kuramitsu HK (June 1997)."Identification of essential amino acids in the Streptococcus mutans glucosyltransferases".Journal of Bacteriology.179(11): 3391–6.doi:10.1128/jb.179.11.3391-3396.1997.PMC179127.PMID9171379.
  8. ^abMonchois V, Willemot RM, Monsan P (April 1999)."Glucansucrases: mechanism of action and structure-function relationships".FEMS Microbiology Reviews.23(2): 131–51.doi:10.1111/j.1574-6976.1999.tb00394.x.PMID10234842.
  9. ^van Hijum SA, Kralj S, Ozimek LK, Dijkhuizen L, van Geel-Schutten IG (March 2006)."Structure-function relationships of glucansucrase and fructansucrase enzymes from lactic acid bacteria".Microbiology and Molecular Biology Reviews.70(1): 157–76.doi:10.1128/MMBR.70.1.157-176.2006.PMC1393251.PMID16524921.
  10. ^Robyt JF, Yoon SH, Mukerjea R (December 2008). "Dextransucrase and the mechanism for dextran biosynthesis".Carbohydrate Research.343(18): 3039–48.doi:10.1016/j.carres.2008.09.012.PMID18922515.
  11. ^Jensen MH, Mirza O, Albenne C, Remaud-Simeon M, Monsan P, Gajhede M, Skov LK (March 2004). "Crystal structure of the covalent intermediate of amylosucrase from Neisseria polysaccharea".Biochemistry.43(11): 3104–10.doi:10.1021/bi0357762.PMID15023061.
  12. ^"Dental Researchers to Mouth Bacteria: Don't Get too Attached".2010-12-08. Archived fromthe originalon 2010-12-14.Retrieved2014-02-28.
  13. ^"Finding a Cure for Tooth Decay".2011-05-12.
  14. ^Featherstone JD (September 2008). "Dental caries: a dynamic disease process".Australian Dental Journal.53(3): 286–91.doi:10.1111/j.1834-7819.2008.00064.x.PMID18782377.
  15. ^Baron, S.; Loesche, W. J. (1996)."Microbiology of Dental Decay and Periodontal Disease".Medical Microbiology.University of Texas Medical Branch at Galveston.ISBN978-0-9631172-1-2.PMID21413316.
  16. ^Colby SM, McLaughlin RE, Ferretti JJ, Russell RR (February 1999). "Effect of inactivation of gtf genes on adherence of Streptococcus downei".Oral Microbiology and Immunology.14(1): 27–32.doi:10.1034/j.1399-302x.1999.140103.x.PMID10204477.
  17. ^Soetaert W, Schwengers D, Buchholz K, Vandamme EJ (January 1995). "A wide range of carbohydrate modifications by a single micro-organism: leuconostoc mesenteroides.".Progress in Biotechnology.Vol. 10. Elsevier. pp. 351–358.doi:10.1016/S0921-0423(06)80116-4.ISBN978-0-444-82223-9.
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