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= N-Acetyl-β-d-Glucosaminidase =

N-acetyl-β-d-glucosaminidase (NAGase) is a sugar degrading hexosaminidase enzyme involved in the digestion of chitin. It belongs to the clan K, family 20 glycosyl hydrolases, and works as an endoenzyme chitinase on oligosaccharides.

Biological Function
In the marine environment, chitin is one of the most prevalent carbohydrates, with yearly estimates of biosynthesis from 1010 – 1011 million tons. It is found throughout the phylogenetic tree in almost all animal taxa.

Generally, crustaceans are the organisms associated with chitin, and a number of studies have been done observing N-acetyl-β-d-glucosaminidase in Antarctic krill (Euphausia superba). In these organisms, researchers have actually found two forms of the enzyme; NAGase B and NAGase C. In E. superba, NAGase B is active in the molt cycle and is part of the integument system. NAGase B is a significant component of chitin degradation while the krill is molting. NAGase B’s catalysis of N-acetyl glucosamine monomers causes a 50% decrease in chitin content and allows the resorption of chitin through the epidermis and further metabolic processing – mostly the development of a new cuticle – during the molting process. Alternatively, NAGase C is released in the gastrointestinal system and is part of the digestive process when E. superba consumes chitin containing organisms.

N-acetyl-β-d-glucosaminidase has also been present in the digestive systems of fish. Crustaceans are a major component of the bonnethead shark’s (Sphyrna tiburo) diet, and chitin is thought to be a significant source of carbon and nitrogen for bonnethead and other sharks. For these and other fish, studies have shown that the majority of digestive chitinolytic enzyme activity originates in the stomach, intestines and hind-gut. “Chitinases hydrolyze the chitin polysaccharides into insoluble dimers or trimers” in part to break down the skeletal structure of the fish, but also to aid other digestive enzyme’s infiltration of their prey’s tissue.

Crystal Structure & How it Determines Function
This enzyme is a homodimer, with each subunit made up of three domains. It has two crystal structures (I and II). Crystal structure I has two subunits in the asymmetric unit, while II only has one.

“The main difference between the two crystal forms involves the conformation of the β-strand containing the catalytic acids (D223 and E224) and the β-strand and βα-loop directly adjacent to these acids. In both subunits of the form I structure, the catalytic acids are positioned above the tryptophan-lined pocket, as if poised for catalysis. In the form II structure, conformational changes relocate the side-chain of E224 away from the tryptophan-lined pocket, which appears to be larger and more exposed to solvent."

Research has suggested that structure I is the active form, while structure II is inactive.

Active Sites
The active sites for N-acetyl-β-d-glucosaminidase are located in the TIM-barrel domain of the structure. They are at the bottom of cavities on the protein subunits of the individual dimers. The two active sites are connected by a solvent channel. The active site cavities where the terminal carbohydrates bind are lined by “four tryptophan residues and is adjacent to the two conserved acids (D223 and E224).”

Reaction Pathway
N-acetyl-β-d-glucosaminidase is a Nexochitinase subcategory chitobiase. It acts on both N-acetylglucosides and N-acetylgalactosides. It works with endochitinates to split the chitin chain into in to oligosaccharides. “They synergistically and consecutively hydrolyze the polysaccharide to monomers of N-acetyl-glucosamine. Chitinase hydrolyzes chitin chains into trimers and dimers while chitobiase further hydrolyzes the smaller units into N-acetyl-glucosamine monomers".

Function in the Cell
The degradation of chitin is a multi-step process. The pathway involving N-acetyl-β-d-glucosaminidase has the chitobiosinization of chitin to chitosan. Chisosanase hydrolyzes the glycosidic bonds to generate glucosaminylglucosaminide which N-acetyl-β-d-glucosaminidase will then hydrolyze in to glucosamine.