Processivity and substrate-binding in family 18 chitinases

Sørlie, Morten; Zakariassen, Henrik; Norberg, Anne Line; Eijsink, Vincent G. H.

doi:10.3109/10242422.2012.676282

Cited by 20 publications

(33 citation statements)

References 89 publications

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“…Though the processive mechanism of GHs has been extensively studied by both biochemical and structural studies [8,13**,14**, 18,30,32,33,37,39,[41][42][43][44][45][46][47][48][49][50], the direct connection of structure to processive function beyond the broad topological categorizations remains unclear. We recently described the hypothesized elementary steps involved in the catalytic cycle of a processive GH as a free energy profile along the enzymatic reaction coordinate [51].…”

Section: Thermodynamics Of Cbh Processivitymentioning

confidence: 99%

Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases

Beckham

Ståhlberg

Knott

et al. 2014

Current Opinion in Biotechnology

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Polysaccharide depolymerization in nature is primarily accomplished by processive glycoside hydrolases (GHs), which abstract single carbohydrate chains from polymer crystals and cleave glycosidic linkages without dissociating after each catalytic event.Understanding the molecular-level features and structural aspects of processivity is of importance due to the prevalence of processive GHs in biomass-degrading enzyme cocktails. Here, we describe recent advances towards the development of a molecular-level theory of processivity for cellulolytic and chitinolytic enzymes, including the development of novel methods for measuring rates of key steps in processive action and insights gained from structural and computational studies. Overall, we present a framework for developing structure-function relationships in processive GHs and outline additional progress towards developing a fundamental understanding of these industrially important enzymes. IntroductionStructural polysaccharides, such as cellulose and chitin, typically arrange in insoluble, polymeric crystals that form significant components of plant, fungal, and algal cell walls. Microorganisms have evolved suites of enzymatic machinery to degrade these polysaccharides to soluble units for food and energy. These enzyme cocktails are primarily composed of various glycoside hydrolases (GHs) with synergistic functions to efficiently cleave the glycosidic linkages [1,2]. More recently, additional enzymatic functions beyond the canonical GH enzyme battery have been discovered including oxidative enzymes that selectively cleave glycosidic bonds [3][4][5][6][7]. GH cocktails contain enzymes typically delineated into two broadly defined classes: cellobiohydrolases (CBHs) and endoglucanases (EGs) for cellulose depolymerization, or chitobiohydrolases and endochitinases for chitin depolymerization. EGs are thought to randomly hydrolyze glycosidic linkages primarily in amorphous regions of polymer fibers. Alternatively, CBHs are able to attach to carbohydrate chains and processively hydrolyze disaccharide units from the end of a chain without dissociation after each catalytic event. Processivity is traditionally thought to be a means of conserving energy during enzymatic function, and is a general strategy used in the synthesis, modification, and depolymerization of many natural biopolymers [8]. It is this ability to act processively that imparts significant hydrolytic potential to CBHs from various GH families such as GH Family 6, 7, 18, and 48 and typically makes them the most abundant enzymes in natural secretomes of many microorganisms. Thus, GHs are the focus of intense protein engineering efforts for the biofuels industry [9*,10].

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Section: Thermodynamics Of Cbh Processivitymentioning

confidence: 99%

Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases

Beckham

Ståhlberg

Knott

et al. 2014

Current Opinion in Biotechnology

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“…Sliding dynamics are displayed by the chitinases grouped as family 18 14. These enzymes have chitin‐binding grooves (Figure 2 a) and depolymerize their substrates, and is of great interest for biomass refinement.…”

Section: Processive Enzymes In Naturementioning

confidence: 99%

“… Chitinase and its substrate‐binding groove 14. A) Two views of chitinase B from S. marcescens (PDB: 1E15).…”

Section: Processive Enzymes In Naturementioning

confidence: 99%

Processive Catalysis

et al. 2014

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Nature's enzymes are an ongoing source of inspiration for scientists. The complex processes behind their selectivity and efficiency is slowly being unraveled, and these findings have spawned many biomimetic catalysts. However, nearly all focus on the conversion of small molecular substrates. Nature itself is replete with inventive catalytic systems which modify, replicate, or decompose entire polymers, often in a processive fashion. Such processivity can, for example, enhance the rate of catalysis by clamping to the polymer substrate, which imparts a large effective molarity. Reviewed herein are the various strategies for processivity in nature's arsenal and their properties. An overview of what has been achieved by chemists aiming to mimic one of nature's greatest tricks is also included.

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“…Usually, processive chitinases have a deep catalytic cleft and a path of aromatic amino acid residues from the chitin-binding domain to the catalytic domain. This path helps in the correct positioning of substrate on the catalytic subsites, and the hydrophobic interactions give a strong but flexible binding to guide the chitin chain into the active site (1,7). …”

Section: Introductionmentioning

confidence: 99%

A broad pH range and processive chitinase from a metagenome library

Thimoteo

Glogauer

Faoro

et al. 2017

Braz J Med Biol Res

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Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

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Processivity and substrate-binding in family 18 chitinases

Cited by 20 publications

References 89 publications

Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases

Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases

Processive Catalysis

A broad pH range and processive chitinase from a metagenome library

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