Dispersin B

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Dispersin B
DispB.jpeg
Ribbon diagram of Dispersin B
Identifiers
EC no. 3.2.1.52
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Dispersin B (also known as DspB or DispersinB) is a 40 kDa glycoside hydrolase produced by the periodontal pathogen, Aggregatibacter actinomycetemcomitans . [1] The bacteria secrete Dispersin B to release adherent cells from a mature biofilm colony by disrupting biofilm formation. The enzyme catalyzes the hydrolysis of linear polymers of N-acetyl-D-glucosamines found in the biofilm matrices. Poly-acetyl glucosamines are integral to the structural integrity of the biofilms of various Gram-positive bacteria and Gram-negative bacteria and are referred to as PIA (PNAG,PS/A) in Staphylococcus species and PGA in Escherichia coli . [2] [3] By degrading the biofilm matrix, Dispersin B allows for the release of bacterial cells that can adhere to new surfaces close by and extend the biofilm or start new colonies. Currently there is interest in Dispersin B as a commercial anti-biofilm agent that could be combined with antibiotics for the treatment of bacterial infections.

Contents

Structure of Poly-N-acetyl-D-glucosamine PNAG.png
Structure of Poly-N-acetyl-D-glucosamine

Biological function

Dispersin B is produced by Aggregatibacter actinomycetemcomitans, a Gram-negative oral bacterium, when it needs to detach and disperse adherent bacterial cells. [4] A. actinomycetemcomitans forms asymmetric biofilm lobed colonies that release single cells or small clusters of bacterial cells, which can attach to nearby surfaces, form new colonies, and enable the biofilm to spread. [2] [5] A biofilm is a matrix of extracellular polymeric substances that are synthesized by the bacteria. The biofilm's structural integrity is dependent on poly-N-acetylglucosamine (PGA), extracellular DNA, and proteinaceous adhesins. It allows bacteria to adhere to host surfaces, protects the bacterial cells from host defenses, results in increased resistance to antibiotics, and provides a protected environment with microchannels for the flow of water and other essential nutrients. [2] By hydrolyzing PGA, Dispersin B disrupts the formation of the biofilm matrix and allows adherent cells to be released. [6] Dispersin B has also been shown to cause the detachment of biofilm cells that have adhered to abiotic surfaces as well as cause the disaggregaton of highly auto-aggregated clumps of bacterial cells. [2]

Enzyme structure

Single Domain of Dispersin B: TIM Barrel (major substructure) shown in blue. a-Helices are represented by cylinders and b-strands represented by arrows Single Domain of Dispersin B.png
Single Domain of Dispersin B: TIM Barrel (major substructure) shown in blue. α-Helices are represented by cylinders and β-strands represented by arrows

The three-dimensional structure of dispersin B was determined by expressing the protein in E. coli, purifying it from cultures, and crystallizing it in the orthoorhombic space group C2221 with one molecule in the asymmetric unit. [2] It is a 361 amino acid protein consisting of a single domain. The major substructure consists of residues 1-60 and residues 91-358, which fold into a β/α structure of a typical TIM barrel. The loop β4 contains two highly conserved residues Asp183 and Glu184 that are part of the enzyme's active site, a large cavity in the center of the bowl-shaped Dispersin B molecule. The oligomer substrate binding and cleaving at the active site of Dispersin B was studied to learn more about the enzymatic degradation. [7] Reverse phase HPLC was used to analyze the products of the hydrolysis of substrates with differing degrees of polymerization. Chain lengthening of the substrate was shown to increase the catalytic efficiency of Dispersin B. A substrate with a degree of polymerization of five was not enough for total subsite occupancy. [7] Carbamate substrates were also shown to improve the activity of the enzyme relative to the benchmark substrate 4-nitrophenyl N-acetyl-β-D-glucosamine. [8]

Enzyme mechanism

Dispersin B is a β-hexosaminidase that specifically hydrolyzes β-1,6-glycosidic linkages of acetylglucosamine polymers found in biofilm matrices. [9] As a member of family 20 β-hexosaminidases, it cleaves terminal monosaccharide residues from the non-reducing end of the polymers. The active site of Dispersin B contains three highly conserved acidic residues: an aspartic acid at residue 183 (D183), a glutamic acid at residue 184 (E184), and a glutamic acid at residue 332 (E332). [10] In the proposed mechanism, E184 serves as the acid/base and donates a proton to the -OR on C1. Crystallographic evidence suggests that substrate-assisted catalysis occurs and D183 is believed to assist in activating the N-acetyl group, which acts as a nucleophilic in an attack of C1. [11] Water then attacks the anomeric centre to release the cleaved polymer from the active site with retention of anomeric configuration. (The mechanism shown below is thus incorrect). [10] [12] The use of the neighboring C2-acetamido group of the substrate differs from the majority of β-glycosidases, which use a carboxyl group as a nucleophile in their mechanisms. [13] [14] Although E332 is located farther away from the anomeric carbon, the lower activity of Dispersin B with the mutation E332Q suggests that it is important for catalysis. It may be critical in stabilizing the transition state of cleaving the terminal monosaccharide. [15]

Active Site of Dispersin B: E184 shown in blue. D183 shown in pink. E332 shown in yellow. Active Site of Dispersin B.png
Active Site of Dispersin B: E184 shown in blue. D183 shown in pink. E332 shown in yellow.
Proposed Mechanism of Dispersin B: E184 shown in blue. D183 shown in pink. E332 not shown. DispersinB Mechanism.png
Proposed Mechanism of Dispersin B: E184 shown in blue. D183 shown in pink. E332 not shown.

Applications

Dispersin B may be useful for treating and preventing biofilm-associated infections caused by poly-N-acetylglucosamine-producing bacteria. Dispersin B prevented the formation of S. epidermidis biofilm, which suggests that biofilm-releasing enzymes can exhibit broad-spectrum activity and could be potential antibiofilm agents. [16]

Commercial development

Dispersin B is being commercially developed as a wound care gel and medical device coating by Kane Biotech, Inc., a Canadian biotech company. External and internal catheters that were coated with a combination of Dispersin B and triclosan were shown to be as effective at preventing bacterial infection as already commercially available chlorhexidine-sulfadiazine-coated catheters. These findings support that Dispersin B exhibits synergistic activity when combined with antibiotics. [7] Carbamate substrates were also shown to improve the activity of the enzyme relative to the benchmark substrate 4-nitrophenyl N-acetyl-β-D-glucosamine. [17] Pharmaceutical-grade Dispersin B has been successfully manufactured by BioVectra, Inc., Charlottetown, P.E.I., Canada, and is currently undergoing biocompatibility testing. Clinical trials to test the effectiveness of dispersin B for the treatment and prevention of diabetic foot ulcers and pressure sores should begin in 2010.

Related Research Articles

Peptidoglycan or murein is a unique large macromolecule, a polysaccharide, consisting of sugars and amino acids that forms a mesh-like peptidoglycan layer (sacculus) that surrounds the bacterial cytoplasmic membrane. The sugar component consists of alternating residues of β-(1,4) linked N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). Attached to the N-acetylmuramic acid is an oligopeptide chain made of three to five amino acids. The peptide chain can be cross-linked to the peptide chain of another strand forming the 3D mesh-like layer. Peptidoglycan serves a structural role in the bacterial cell wall, giving structural strength, as well as counteracting the osmotic pressure of the cytoplasm. This repetitive linking results in a dense peptidoglycan layer which is critical for maintaining cell form and withstanding high osmotic pressures, and it is regularly replaced by peptidoglycan production. Peptidoglycan hydrolysis and synthesis are two processes that must occur in order for cells to grow and multiply, a technique carried out in three stages: clipping of current material, insertion of new material, and re-crosslinking of existing material to new material.

<span class="mw-page-title-main">Biofilm</span> Aggregation of bacteria or cells on a surface

A biofilm is an syntrophic community of microorganisms in which cells stick to each other and often also to a surface. These adherent cells become embedded within a slimy extracellular matrix that is composed of extracellular polymeric substances (EPSs). The cells within the biofilm produce the EPS components, which are typically a polymeric combination of extracellular polysaccharides, proteins, lipids and DNA. Because they have three-dimensional structure and represent a community lifestyle for microorganisms, they have been metaphorically described as "cities for microbes".

<span class="mw-page-title-main">Teichoic acid</span>

Teichoic acids are bacterial copolymers of glycerol phosphate or ribitol phosphate and carbohydrates linked via phosphodiester bonds.

<i>N</i>-Acetylglucosamine Biological molecule

N-Acetylglucosamine (GlcNAc) is an amide derivative of the monosaccharide glucose. It is a secondary amide between glucosamine and acetic acid. It is significant in several biological systems.

The terms glycans and polysaccharides are defined by IUPAC as synonyms meaning "compounds consisting of a large number of monosaccharides linked glycosidically". However, in practice the term glycan may also be used to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan, even if the carbohydrate is only an oligosaccharide. Glycans usually consist solely of O-glycosidic linkages of monosaccharides. For example, cellulose is a glycan composed of β-1,4-linked D-glucose, and chitin is a glycan composed of β-1,4-linked N-acetyl-D-glucosamine. Glycans can be homo- or heteropolymers of monosaccharide residues, and can be linear or branched.

<span class="mw-page-title-main">Hexosaminidase</span> Class of enzymes

Hexosaminidase is an enzyme involved in the hydrolysis of terminal N-acetyl-D-hexosamine residues in N-acetyl-β-D-hexosaminides.

<span class="mw-page-title-main">N-acetylglucosamine-6-sulfatase</span> Protein-coding gene in the species Homo sapiens

N-acetylglucosamine-6-sulfatase (EC 3.1.6.14, glucosamine (N-acetyl)-6-sulfatase, systematic name N-acetyl-D-glucosamine-6-sulfate 6-sulfohydrolase) is an enzyme that in humans is encoded by the GNS gene. It is deficient in Sanfilippo Syndrome type IIId. It catalyses the hydrolysis of the 6-sulfate groups of the N-acetyl-D-glucosamine 6-sulfate units of heparan sulfate and keratan sulfate

The enzyme mannosyl-glycoprotein endo-β-N-acetylglucosaminidase (endoglycosidase H) (EC 3.2.1.96) has systematic name glycopeptide-D-mannosyl-N4-(N-acetyl-D-glucosaminyl)2-asparagine 1,4-N-acetyl-β-glucosaminohydrolase. It is a highly specific endoglycosidase which cleaves asparagine-linked mannose rich oligosaccharides, but not highly processed complex oligosaccharides from glycoproteins. It is used for research purposes to deglycosylate glycoproteins and to monitor intracellular protein trafficking through the secretory pathway.

Aggregatibacter actinomycetemcomitans is a Gram-negative, facultative anaerobe, nonmotile bacterium that is often found in association with localized aggressive periodontitis, a severe infection of the periodontium. It is also suspected to be involved in chronic periodontitis. Less frequently, A. actinomycetemcomitans is associated with nonoral infections such as endocarditis. Its role in aggressive periodontitis was first discovered by Danish-born periodontist Jørgen Slots, a professor of dentistry and microbiology at the University of Southern California School of Dentistry.

<span class="mw-page-title-main">N-acetylglucosamine-6-phosphate deacetylase</span>

In enzymology, N-acetylglucosamine-6-phosphate deacetylase (EC 3.5.1.25), also known as GlcNAc-6-phosphate deacetylase or NagA, is an enzyme that catalyzes the deacetylation of N-acetylglucosamine-6-phosphate (GlcNAc-6-P) to glucosamine-6-phosphate (GlcN-6-P):

In enzymology, a glucosamine-1-phosphate N-acetyltransferase is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Glucosamine-phosphate N-acetyltransferase</span>

In enzymology, glucosamine-phosphate N-acetyltransferase (GNA) is an enzyme that catalyzes the transfer of an acetyl group from acetyl-CoA to the primary amine in glucosamide-6-phosphate, generating a free CoA and N-acetyl-D-glucosamine-6-phosphate.

<span class="mw-page-title-main">UDP-N-acetylglucosamine 1-carboxyvinyltransferase</span> Class of enzymes

In enzymology, an UDP-N-acetylglucosamine 1-carboxyvinyltransferase is an enzyme that catalyzes the first committed step in peptidoglycan biosynthesis of bacteria:

In enzymology, an undecaprenyldiphospho-muramoylpentapeptide beta-N-acetylglucosaminyltransferase is an enzyme that catalyzes the chemical reaction

Protein <i>O</i>-GlcNAc transferase Protein-coding gene in the species Homo sapiens

Protein O-GlcNAc transferase also known as OGT or O-linked N-acetylglucosaminyltransferase is an enzyme that in humans is encoded by the OGT gene. OGT catalyzes the addition of the O-GlcNAc post-translational modification to proteins.

UDP-N-acetylglucosamine kinase is an enzyme with systematic name ATP:UDP-N-acetyl-alpha-D-glucosamine 3'-phosphotransferase. This enzyme catalyses the following chemical reaction

UDP-N-acetylglucosamine—undecaprenyl-phosphate N-acetylglucosaminephosphotransferase is an enzyme with systematic name UDP-N-acetyl-alpha-D-glucosamine:ditrans,octacis-undecaprenyl phosphate N-acetyl-alpha-D-glucosaminephosphotransferase. This enzyme catalyses the following chemical reaction

Protein <i>O</i>-GlcNAcase Protein-coding gene in the species Homo sapiens

Protein O-GlcNAcase (EC 3.2.1.169, OGA, glycoside hydrolase O-GlcNAcase, O-GlcNAcase, BtGH84, O-GlcNAc hydrolase) is an enzyme with systematic name (protein)-3-O-(N-acetyl-D-glucosaminyl)-L-serine/threonine N-acetylglucosaminyl hydrolase. OGA is encoded by the OGA gene. This enzyme catalyses the removal of the O-GlcNAc post-translational modification in the following chemical reaction:

  1. [protein]-3-O-(N-acetyl-β-D-glucosaminyl)-L-serine + H2O ⇌ [protein]-L-serine + N-acetyl-D-glucosamine
  2. [protein]-3-O-(N-acetyl-β-D-glucosaminyl)-L-threonine + H2O ⇌ [protein]-L-threonine + N-acetyl-D-glucosamine
<span class="mw-page-title-main">Epimerox</span> Chemical compound

Epimerox is an experimental broad-spectrum antibiotic compound being developed by scientists at the Rockefeller University and Astex Pharmaceuticals. It is a small molecule inhibitor compound that blocks the activity of the enzyme UDP-N-acetylglucosamine 2-epimerase, an epimerase enzyme that is called 2-epimerase for short.

N-acetyl-β-d-glucosaminidase(EC 3.2.1.30; EC 3.2.1.52) is a mesophilic hydrolase that specifically hydrolyzes N-acetyl-glucosides. The enzyme is found across a wide variety of marine and terrestrial creatures with the primary function of breaking down oligosaccharides in the presence of water. One of the primary functions of the enzyme is to target and hydrolyze oligosaccharides containing chitin. In this chitinase function, the enzyme contributes to the ability of many organisms to break down chitin-containing molecules and subsequently digest or re-uptake environmental chitin, carbon, or nitrogen. The enzyme's crystal structure varies slightly across organisms, but is characterized by three or four domains with one active site. Across proteins, the active site entails an α-β barrel with either an arginine or tryptophan residues in the barrel pocket to bind incoming substrate.

References

  1. ↑ Kaplan JB, Ragunath C, Ramasubbu N, Fine DH (August 2003). "Detachment of Actinobacillus actinomycetemcomitans biofilm cells by an endogenous beta-hexosaminidase activity". J. Bacteriol. 185 (16): 4693–8. doi:10.1128/JB.185.16.4693-4698.2003. PMC   166467 . PMID   12896987.
  2. 1 2 3 4 5 PDB: 1YHT ​; Ramasubbu N, Thomas LM, Ragunath C, Kaplan JB (June 2005). "Structural analysis of dispersin B, a biofilm-releasing glycoside hydrolase from the periodontopathogen Actinobacillus actinomycetemcomitans". J. Mol. Biol. 349 (3): 475–86. doi:10.1016/j.jmb.2005.03.082. PMID   15878175.
  3. ↑ Mack D, Fischer W, Krokotsch A, Leopold K, Hartmann R, Egge H, Laufs R (1996). "The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear beta-1,6-linked glucosaminoglycan: purification and structural analysis". J. Bacteriol. 178 (1): 175–183. PMC   177636 . PMID   8550413.
  4. ↑ Blackledge MS, Worthington RJ, Melander C (2013). "Biologically inspired strategies for combating bacterial biofilms". Curr Opin Pharmacol. 13 (5): 699–706. doi:10.1016/j.coph.2013.07.004. PMC   3795836 . PMID   23871261.
  5. ↑ Kaplan JB, Meyenhofer MF, Fine DH (2003). "Biofilm growth and detachment of Actinobacillus actinomycetemcomitans". J. Bacteriol. 185 (4): 1399–1404. doi:10.1128/JB.185.4.1399-1404.2003. PMC   142852 . PMID   12562811.
  6. ↑ Izano EA, Sadovskaya I, Vinogradov E, Mulks MH, Velliyagounder K, Ragunath C, Kher WB, Ramasubbu N, Jabbouri S, Perry MB, Kaplan JB (2007). "Poly-N-acetylglucosamine mediates biofilm formation and antibiotic resistance in Actinobacillus pleuropneumoniae". Microb Pathog. 43 (1): 1–9. doi:10.1016/j.micpath.2007.02.004. PMC   1950449 . PMID   17412552.
  7. 1 2 3 Fazekas E, Kandra L, Gyémánt G (2012). "Model for β-1,6-N-acetylglucosamine oligomer hydrolysis catalysed by DispersinB, a biofilm degrading enzyme". Carbohydrate Res. 363: 7–13. doi:10.1016/j.carres.2012.09.016. PMID   23103508.
  8. ↑ Chibba A, Dasgupta S, Yakandawala N, Madhyastha S, Nitz M (2011). "Chromogenic Carbamate and Acetal Substrates for Glycosaminidases". J. Carbohydr. Chem. 30: 549–558. doi:10.1080/07328303.2011.610543. ISSN   0732-8303.
  9. ↑ Itoh Y, Wang X, Hinnebusch BJ, Preston JF, Romeo T (2005). "Depolymerization of beta-1,6-N-acetyl-D-glucosamine disrupts the integrity of diverse bacterial biofilms". J. Bacteriol. 187 (1): 382–386. doi:10.1128/JB.187.1.382-387.2005. PMC   538831 . PMID   15601723.
  10. 1 2 Manuel SG, Ragunath C, Sait HB, Izano EA, Kaplan JB, Ramasubbu N (2007). "Role of active-site residues of dispersin B, a biofilm-releasing beta-hexosaminidase from a periodontal pathogen, in substrate hydrolysis". FEBS J. 274 (22): 5987–99. doi: 10.1111/j.1742-4658.2007.06121.x . PMID   17949435.
  11. ↑ Mark BL, Vocadlo DJ, Knapp S, Triggs-Raine BL, Withers SG, James MN (2001). "Crystallographic evidence for substrate-assisted catalysis in a bacterial beta-hexosaminidase". J Biol Chem. 276 (13): 10330–10337. doi: 10.1074/jbc.M011067200 . PMID   11124970.
  12. ↑ Hou Y, Vocadlo DJ, Leung A, Withers SG, Mahuran D (2001). "Characterization of the Glu and Asp residues in the active site of human beta-hexosaminidase B". Biochemistry. 40 (7): 2201–2209. doi:10.1021/bi002018s. PMC   2910085 . PMID   11329289.
  13. ↑ Maier T, Strater N, Schuette CG, Klingenstein R, Sandhoff K, Saenger W (2003). "The X-ray crystal structure of human beta-hexosaminidase B provides new insights into Sandhoff disease". J Mol Biol. 328 (3): 669–681. doi:10.1016/s0022-2836(03)00311-5. PMID   12706724.
  14. ↑ Drouillard S, Armand S, Davies GJ, Vorgias CE, Henrissat B (1997). "hem J. 1997 Dec 15;328 ( Pt 3):945-9. Serratia marcescens chitobiase is a retaining glycosidase utilizing substrate acetamido group participation". Biochem. J. 328 (3): 945–949. doi:10.1042/bj3280945. PMC   1219008 . PMID   9396742.
  15. ↑ Williams SJ, Mark BL, Vocadlo DJ, James MN, Withers SG (2002). "Aspartate 313 in the Streptomyces plicatus hexosaminidase plays a critical role in substrate-assisted catalysis by orienting the 2-acetamido group and stabilizing the transition state". J Biol Chem. 277 (42): 40055–40065. doi: 10.1074/jbc.M206481200 . PMID   12171933.
  16. ↑ Kaplan JB, Ragunath C, Velliyagounder K, Fine DH, Ramasubbu N (2004). "Enzymatic detachment of Staphylococcus epidermidis biofilms". Antimicrob. Agents Chemother. 48 (7): 2633–2636. doi:10.1128/AAC.48.7.2633-2636.2004. PMC   434209 . PMID   15215120.
  17. ↑ Darouiche RO, Mansouri MD, Gawande PV, Madhyastha S (2009). "Antimicrobial and antibiofilm efficacy of triclosan and DispersinB combination". Journal of Antimicrobial Chemotherapy. 64 (1): 88–93. doi: 10.1093/jac/dkp158 . PMID   19447791.
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