N-Acetyl-β-d-Glucosaminidase

N-acetyl-β-d-glucosaminidase (NAGase) is a sugar degrading hexosaminidase enzyme involved in the digestion of chitin^[1]. It belongs to the clan K, family 20 glycosyl hydrolases^[2][3], and works as an endoenzyme chitinase on oligosaccharides^[4].

Biological Function

In the marine environment, chitin is one of the most prevalent carbohydrates, with yearly estimates of biosynthesis from 10¹⁰ – 10¹¹ million tons^[5]. It is found throughout the phylogenetic tree in almost all animal taxa^[6].

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^[4][6]. In E. superba, NAGase B is active in the molt cycle and is part of the integument system^[4][6][7].  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^[8] and allows the resorption of chitin through the epidermis and further metabolic processing – mostly the development of a new cuticle – during the molting process^[4][6][9]. Alternatively, NAGase C is released in the gastrointestinal system and is part of the digestive process when E. superba consumes chitin containing organisms^{[4][6][7][10]}.

N-acetyl-β-d-glucosaminidase has also been present in the digestive systems of fish^[1][5][11]. 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^[11]. For these and other fish, studies have shown that the majority of digestive chitinolytic enzyme activity originates in the stomach, intestines and hind-gut^[5][11]. “Chitinases hydrolyze the chitin polysaccharides into insoluble dimers or trimers”^[5] 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.^[5][12]

Crystal Structure & How it Determines Function

This enzyme is a homodimer, with each subunit made up of three domains^[2][12]. It has two crystal structures (I and II). Crystal structure I has two subunits in the asymmetric unit, while II only has one^[2].

“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."^[2]

Research has suggested that structure I is the active form, while structure II is inactive^[2].

Active Sites

The active sites for N-acetyl-β-d-glucosaminidase are located in the TIM-barrel domain of the structure^[2]. They are at the bottom of cavities on the protein subunits of the individual dimers^[2]. The two active sites are connected by a solvent channel^[2]. 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).”^[2]

Reaction Pathway

N-acetyl-β-d-glucosaminidase is a Nexochitinase subcategory chitobiase^[3]. It acts on both N-acetylglucosides and N-acetylgalactosides^[1][4][6]. It works with endochitinates to split the chitin chain into in to oligosaccharides^{[1][2][5][12]}. “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"^[5].

Function in the Cell

The degradation of chitin is a multi-step process^[5][12]. 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^[12].

References