Thermodynamic Relationships with Processivity in <i>Serratia marcescens</i> Family 18 Chitinases

Hamre, Anne Grethe; Jana, Suvamay; Holen, Matilde Mengkrog; Mathiesen, Geir; Väljamaë, Priit; Payne, Christina M.; Sørlie, Morten

doi:10.1021/acs.jpcb.5b03817

Cited by 20 publications

(31 citation statements)

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“…3, and Table 1) with K d s of 31~66 nM, 15~30 higher affinities than those of maltoseand galactose-binding proteins (19). ITC analysis of (GlcNAc) n binding were thoroughly conducted for GH18 chitinase B from Serratia marcescens and a GH19 chitinase from Bryum coronatum (20,21), both of which have a long-extended binding cleft for (GlcNAc) n . In both cases, the longer the chain length of (GlcNAc) n , the higher the favorable free energy changes of binding (G˚).…”

Section: Discussionmentioning

confidence: 99%

Structure and function of a novel periplasmic chitooligosaccharide-binding protein from marine Vibrio bacteria

Suginta

Sritho

Ranok

et al. 2018

Journal of Biological Chemistry

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Periplasmic solute-binding proteins (SBPs) in bacteria are involved in the active transport of nutrients into the cytoplasm. In marine bacteria of the genus Vibrio, a chitooligosaccharide-binding protein (CBP) is thought to be the major SBP controlling the rate of chitin uptake in these bacteria. However, the molecular mechanism of the CBP involvement in chitin metabolism has not been elucidated. Here, we report the structure and function of a recombinant chitooligosaccharide-binding protein from Vibrio harveyi, namely VhCBP, expressed in Escherichia coli. Isothermal titration calorimetry (ITC) revealed that VhCBP strongly binds shorter chitooligosaccharides [(GlcNAc) n , n = 2, 3, and 4] with affinities that are considerably greater than those for glycoside hydrolase family 18 (GH18) and GH19 chitinases, but does not bind longer ones, including insoluble chitin polysaccharides. We also found that VhCBP comprises two domains with flexible linkers and that the domain-domain interface forms the sugar-binding cleft, which is not long extended but forms a small cavity. (GlcNAc) 2 bound to this cavity, apparently triggering a closed conformation of VhCBP. Trp-363 and Trp-513, which stack against the two individual GlcNAc rings, likely make a major contribution to the high affinity of VhCBP for (GlcNAc) 2 . The strong chitobiose binding, followed by the conformational change of VhCBP, may facilitate its interaction with an active-transport system in the inner membrane of Vibrio species.Vibrio harveyi is a Gram-negative bioluminescent marine bacterium found in tropical marine waters, and is a serious pathogen of aquatic fish and invertebrates, including crustacean and zooplankton (1). Chitin, -1,4-linked polysaccharide of N-acetylglucosamine (GlcNAc), is an important marine biomass, which is efficiently converted by Vibrios. Thus, V. harveyi has an efficient chitin degradation system, including a chitinase A (2-5) belonging to glycoside hydrolase family 18 (GH18), a -N-acetylglucosaminidase (6,7) belonging to the GH20 family, and a chitin-oligosaccharide deacetylase (8,9) belonging to carbohydrate esterase family 4 (CE4).GH18 chitinases hydrolyze -1,4-glycosidic linkages of chitin in an endo-splitting mode, producing chitin oligosaccharides (GlcNAc) n (n=2, 3, 4, and http://www.jbc.org/cgi

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Section: Discussionmentioning

confidence: 99%

Structure and function of a novel periplasmic chitooligosaccharide-binding protein from marine Vibrio bacteria

Suginta

Sritho

Ranok

et al. 2018

Journal of Biological Chemistry

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“…In nature, chitin is degraded by glycoside hydrolases (GH) and lytic polysaccharide monooxygenases (LPMO) [25,26]. GHs overcome the thermodynamic penalty of decrystallization by strong binding to a single polysaccharide chain through several surface-exposed aromatic amino acids and by this pulling the chain from the crystal into the active site of the enzymes [27,28]. Moreover, the binding affinity of GHs towards the substrate increases with an increasing chain length of the substrate [29,30].…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, the binding affinity of GHs towards the substrate increases with an increasing chain length of the substrate [29,30]. Typical binding free energy of a chitin active GH on six GlcNAc residues is −9 kcal/mol ( K d = 0.2 μM) [28]. LPMOs achieve decrystallization by activating an oxygen species on its copper-active site that is calculated to be strong enough, oxidize a C-H bond of 110 kcal/mol [31].…”

Section: Discussionmentioning

confidence: 99%

Can we make Chitosan by Enzymatic Deacetylation of Chitin?

et al. 2019

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Chitin, an insoluble linear polymer of β-1,4-N-acetyl-d-glucosamine (GlcNAc; A), can be converted to chitosan, a soluble heteropolymer of GlcNAc and d-glucosamine (GlcN; D) residues, by partial deacetylation. In nature, deacetylation of chitin is catalyzed by enzymes called chitin deacetylases (CDA) and it has been proposed that CDAs could be used to produce chitosan. In this work, we show that CDAs can remove up to approximately 10% of N-acetyl groups from two different (α and β) chitin nanofibers, but cannot produce chitosan.

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“…We calculated the absolute free energies of binding cellopentaose to CBMs for all three families using an enhanced sampling free energy method, FEP/λ-REMD. FEP/λ-REMD is an enhanced sampling free energy methodology developed by Jiang, Hodoscek, and Roux [ 53 ], which we have previously implemented for protein–carbohydrate systems obtaining good agreement with experimental data [ 25 , 54 , 55 ]. For two different systems, the CBM–cellopentaose complex in solvent and solvated cellopentaose, the non-bonded interactions of cellopentaose with the rest of the system were systematically turned off to obtain the change in free energy.…”

Section: Methodsmentioning

confidence: 99%

Cellulose-specific Type B carbohydrate binding modules: understanding oligomeric and non-crystalline substrate recognition mechanisms

Kognole

Payne

2018

Biotechnol Biofuels

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BackgroundEffective enzymatic degradation of crystalline polysaccharides requires a synergistic cocktail of hydrolytic enzymes tailored to the wide-ranging degree of substrate crystallinity. To accomplish this type of targeted carbohydrate recognition, nature produces multi-modular enzymes, having at least one catalytic domain appended to one or more carbohydrate binding modules (CBMs). The Type B CBM categorization encompasses several families (i.e., protein folds) of CBMs that are generally thought to selectively bind oligomeric polysaccharides; however, a subset of cellulose-specific CBM families (17 and 28) appear to bind non-crystalline cellulose more tightly than oligomers and in a manner that discriminates between surface topology.ResultsTo provide insight into this unexplained phenomenon, we investigated the molecular-level origins of oligomeric and non-crystalline carbohydrate recognition in cellulose-specific Type B CBMs using molecular dynamics (MD) simulation and free energy calculations. Examining two CBMs from three different families (4, 17, and 28), we describe how protein–ligand dynamics contribute to observed variations in binding affinity of oligomers within the same CBM family. Comparisons across the three CBM families identified factors leading to modified functionality prohibiting competitive binding, despite similarity in sequence and specificity. Using free energy perturbation with Hamiltonian replica exchange MD, we also examined the hypothesis that the open topology of the binding grooves in families 17 and 28 necessitates tight binding of an oligomer, while the more confined family 4 binding groove does not require the same degree of tight binding. Finally, we elucidated the mechanisms of non-crystalline carbohydrate recognition by modeling CBMs complexed with a partially decrystallized cellulose substrate. Molecular simulation provided structural and dynamic data for direct comparison to oligomeric modes of carbohydrate recognition, and umbrella sampling MD was used to determine ligand binding free energy. Comparing both protein–carbohydrate interactions and ligand binding free energies, which were in good agreement with experimental values, we confirmed the hypothesis that family 17 and 28 CBMs bind non-crystalline cellulose and oligomers with different affinities (i.e., high and low).ConclusionsOur study provides an unprecedented level of insight into the complex solid and soluble carbohydrate substrate recognition mechanisms of Type B CBMs, the findings of which hold considerable promise for enhancing lignocellulosic biomass conversion technology and development of plant cell wall probes.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1321-7) contains supplementary material, which is available to authorized users.

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Thermodynamic Relationships with Processivity in Serratia marcescens Family 18 Chitinases

Cited by 20 publications

References 81 publications

Structure and function of a novel periplasmic chitooligosaccharide-binding protein from marine Vibrio bacteria

Structure and function of a novel periplasmic chitooligosaccharide-binding protein from marine Vibrio bacteria

Can we make Chitosan by Enzymatic Deacetylation of Chitin?

Cellulose-specific Type B carbohydrate binding modules: understanding oligomeric and non-crystalline substrate recognition mechanisms

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