Glycoside Hydrolase Family 22

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• Author: ^^^Spencer Williams^^^
• Responsible Curator: ^^^David Vocadlo^^^

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Glycoside Hydrolase Family GH22
Clan	none, lysozyme-type fold
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/GH22.html

Substrate specificities

Glycoside hydrolase family 22 contains proteins with two main functions: lysozymes and α-lactalbumin.

Lysozymes catalyse the hydrolysis of (1→4)-β-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan and between N-acetyl-D-glucosamine residues in chitooligosaccharides. Lysozymes are also referred to as muramidases. Lysozymes from family GH22 are classified as c-type lysozymes (c = chicken), to distinguish them from lysozymes of family GH23, which are sometimes referred to as g-type (g = goose) lysozymes. Lysozymes provide a range of functions that are related to their bacteriolytic action. As secretions in milk, saliva, mucus, and tears, and their presence in egg-white, they protect against bacterial infection through their ability to degrade the bacterial cell wall. As intestinal secretions in ruminants such as the cow (Bos taurus) they assist in lysis of commensal gut bacteria, releasing their nutrients for the host. Human lysozyme defects can result in a rare hereditary condition, amyloidosis VIII, in which lysozyme deposits as amyloid.

α-Lactalbumins are auxiliary proteins that bind to and modify the substrate specificity of galactosyltransferase (which in the absence of α-lactalbumin transfers glucose to N-acetylglucosamine), converting it to lactose synthase (i.e. catalyzing transfer to glucose). It is believed that α-lactalbumins evolved at the outset of mammalian evolution, after divergence of mammalian and avian lineages. α-Lactalbumin expression is induced by prolactin, and occurs within the mammary glands.

Kinetics and Mechanism

HEWL operates through a classical Koshland retaining mechanism involving a covalent glycosyl enzyme intermediate [1].

Catalytic Residues

Inspection of complexes of lysozyme with chitooligosaccharides and chemical intuition led to the proposal of Glu35 as a proton donor [2]. Site directed mutagenesis of Glu35 to Gln35 resulted in a complete loss of activity against Micrococcus luteus cell wall [3]. Together these data support the identity of Glu35 as the general acid/base in a classical Koshland retaining mechanism. In an early study Asp52 was highlighted as a catalytic residue, and proposed to play a role in stablizing an oxocarbenium ion intermediate [2]. The Asp52Asn mutant exhibited approximately 5% wild-type lytic ability against Micrococcus luteus cell wall [3]. Asp52 is believed to function as a catalytic nucleophile, as shown by X-ray crystallographic observation of a covalent bond for the 2-fluoroglycosyl enzyme formed on the E35Q mutant of HEWL using N-acetylglucosaminyl-(1,4)-2-deoxy-2-fluoroglycosyl fluoride, and by mass spectrometric observation of a covalent adduct of the same complex [1].

α-Lactalbumins typically lack the conserved catalytic residues present in lysozymes. They possess a conserved Ca²⁺ binding site.

Three-dimensional structures

The first structure of a GH22 member was that of hen egg white lysozyme (HEWL) [4, 5]. In fact, HEWL has a distinguished history as the first enzyme for which atomic resolution X-ray data was reported, and has attracted great interest as it provided the first molecular view of enzyme catalysis.

Family Firsts

First stereochemistry determination

Retention of glycosyl transfer to N-acetylglucosamine and methanol [6].

First catalytic nucleophile identification

Asp52 of hen egg white lysozyme (HEWL), by X-ray crystallography of covalent complex formed with a 2-fluorosugar [1].

First general acid/base residue identification

Glu35 of HEWL proposed on the basis of X-ray structure of a complex with a chitooligosaccharide [2]; the HEWL Glu35Gln mutant displayed a loss of activity against bacterial cell wall [3].

First 3-D structure

Hen egg-white lysozyme (HEWL) was the first glycosidase, and the first enzyme, to have its three-dimensional structure determined by X-ray diffraction techniques [5].

References

1. Vocadlo DJ, Davies GJ, Laine R, and Withers SG. (2001). Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate. Nature. 2001;412(6849):835-8. DOI:10.1038/35090602 |
2. Blake CC, Johnson LN, Mair GA, North AC, Phillips DC, and Sarma VR. (1967). Crystallographic studies of the activity of hen egg-white lysozyme. Proc R Soc Lond B Biol Sci. 1967;167(1009):378-88. DOI:10.1098/rspb.1967.0035 |
3. Malcolm BA, Rosenberg S, Corey MJ, Allen JS, de Baetselier A, and Kirsch JF. (1989). Site-directed mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme. Proc Natl Acad Sci U S A. 1989;86(1):133-7. DOI:10.1073/pnas.86.1.133 |
4. BLAKE CC, FENN RH, NORTH AC, PHILLIPS DC, and POLJAK RJ. (1962). Structure of lysozyme. A Fourier map of the electron density at 6 angstrom resolution obtained by x-ray diffraction. Nature. 1962;196:1173-6. DOI:10.1038/1961173a0 |
5. Blake CC, Koenig DF, Mair GA, North AC, Phillips DC, and Sarma VR. (1965). Structure of hen egg-white lysozyme. A three-dimensional Fourier synthesis at 2 Angstrom resolution. Nature. 1965;206(4986):757-61. DOI:10.1038/206757a0 |

6. Rupley JA, Gates V. Studies on the enzymic activity of lysozyme, II. The hydrolysis and transfer reactions of N-acetylglucosamine oligosaccharides. Proc. Natl. Acad. Sci. U.S.A. 1967; 57(3):496-510. [1] pmcid=PMC335536