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Glycoside Hydrolase Family 22

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Glycoside Hydrolase Family GH22
Clan none, lysozyme-type fold
Mechanism retaining
Active site residues known
CAZy DB link

Substrate specificities

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

Lysozymes are endo-acting enzymes that 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 and are conserved in mammals. Found in secretions such as milk, saliva, mucus, and tears, and in egg-white, lysozymes protect against bacterial infection through their ability to degrade the bacterial cell wall. Lysozymes are also intestinal secretions in the foregut of ruminants including cattle (Bos taurus) and leaf-eating monkeys (e.g. langurs), where it is proposed that they assist in lysis of commensal gut bacteria, releasing their nutrients for the host [1, 2]. Defects in the gene encoding human lysozyme can result in a rare hereditary condition, amyloidosis VIII, in which lysozyme deposits as a protein aggregate [3].

α-Lactalbumins are auxiliary proteins that bind to and modify the substrate specificity of galactosyltransferase (a family GT7 enzyme that in the absence of α-lactalbumin transfers glucose to N-acetylglucosamine), converting it to the heterodimer lactose synthase, which catalyzes transfer to glucose. α-Lactalbumin expression is induced by prolactin, and occurs within the mammary glands. It is believed that α-lactalbumins evolved at the outset of mammalian evolution, after divergence of mammalian and avian lineages [4, 5]. α-Lactalbumins possess a conserved Ca2+ binding site, with a high affinity for the cation of 3-5 nM [6]. α-Lactalbumin possesses no meaningful catalytic activity against peptidoglycan, nor does it bind chitooligosaccharides.

Kinetics and Mechanism

HEWL was the first enzyme for which the 3-dimensional structure was solved [7, 8]. Structural analysis of HEWL in complex with various ligands, as discussed below, led to a proposal that this enzyme uses a catalytic mechanism in which an ion pair intermediate is formed in which a sugar oxocarbenium pairs with an anionic enzymic residue within the active site. The preponderance of data now supports HEWL operating through a classical Koshland retaining mechanism involving a covalent glycosyl enzyme intermediate. Evidence in support of this mechanism was obtained by detection of a covalent glycosyl enzyme intermediate (1) through the use of the substrate chitobiosyl fluoride and the HEWL E35Q (acid/base) mutant, detected by mass spectrometry; (2) use of the modified substrate N-acetylglucosaminyl-(1,4)-2-deoxy-2-fluoroglycosyl fluoride in conjunction with mass spectrometry; and (3) combined use of N-acetylglucosaminyl-(1,4)-2-deoxy-2-fluoroglycosyl fluoride and the HEWL E35Q mutant permitted both detection of a stable intermediate by mass spectrometry and its examination by X-ray crystallography [9]. Notably, a mechanism involving neighboring group participation was ruled out by showing that substrates bearing hydrogen and hydroxyl substitutions at 2-position are hydrolysed at similar rates to that of the parent compound bearing the acetamido group [10, 11], and thus that the 2-acetamido functionality is not critical for catalysis. HEWL catalyzed the hydrolysis of a series of aryl chitobiosides with varying leaving group ability with similar KM values, but varying kcat values, giving support for a rate-determining step involving concerted acid-base or acid-nucleophilic catalysis [12].

HEWL-catalyzed hydrolysis of phenyl chitobioside afforded secondary kinetic isotope effects for substrates isotopologous at the anomeric centre (H1/D1) of kH/kD 1.11, which in combination with the linear free energy analyses, reveals considerable oxocarbenium ion character for the glycosylation transition state of the enzyme-catalyzed reaction [10]. The natural substrate for lysozymes are the bacterial cell wall comprised of alternating NAG and NAM residues. For defined molecular species, maximal rates of lysozyme action occur for (NAG-NAM)3, or the chitin hexasaccharide (NAG)6, demonstrating that the enzyme possesses 6 subsites.

Catalytic Residues

Inspection of complexes of lysozyme with chitooligosaccharides and chemical reasoning led to the proposal of Glu35 as a proton donor [13]. Site directed mutagenesis of Glu35 to Gln35 provided a mutant with no activity against Micrococcus luteus cell wall [14]. 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 as noted above [13]. An early example of unnatural amino acid mutagenesis realized by chemical mutagenesis of Asp52 to Homoser52 yielded an enzyme with greatly reduced catalytic activity [15]. Unexpectedly, the Asp52Asn mutant exhibited approximately 5% wild-type lytic ability against Micrococcus luteus cell wall [14] and this residual activity was shown to arise from the presence of carboxylate groups within the stem peptide of certain peptidoglycan fragments, which presumably act by substrate-assisted catalysis to provide chemical rescue of the mutant [16]. Asp52 is generally now 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 [9].

α-Lactalbumins typically lack the conserved catalytic residues present in lysozymes. Two naturally occurring variants of human lysozyme, Ile56Thr and Asp67His, are amyloidogenic [3]. In both cases, decreased protein stability is believed to contribute to amyloid formation, with fibrils forming more readily at low pH or at slightly elevated temperatures.

Three-dimensional structures

A large number of structures is available for family GH22 members. The bulk of the discussion here will focus on hen egg white lysozyme (HEWL), as it was the first structure reported for a GH22 member [7, 8]. 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, launching the field of structural enzymology. An extensive range of lysozyme structures have been determined, including hundreds of structures of mutants, such that lysozyme is the most commonly deposited protein in the Protein Databank [17]. Lysozyme adopts a compact globular structure comprised of just 127 amino acids. There are five helical regions comprising around 40% of the amino acids. There are also five regions of beta sheet with both random coil and beta turns. A large cleft running across the face of the structure that harbours the active site and the catalytic residues Glu35 and Asp52. Four disulfide bonds are present in the structure: Cys6-Cys127, Cys30-Cys115, Cys64-Cys80, and Cys76-Cys94.

A range of complexes of peptidoglycan derived NAG-NAM oligosaccharides and chitooligosaccharides have been determined, spanning mostly the -4 to -2 (A-C subsites). NAM-NAG-NAM binds in the -3/-2/-1 (B/C/D) subsites and the -1 subsite NAM was described as adopting an envelope conformation [18]; however, interpretation of this complex suffers from considerable disorder manifested as high temperature factors associated with the sugar bound in the -1 subsite [19]. A complex with a chitotetraose-derived lactone, which was proposed to be a transition state analogue by viture of its tight binding, was interpreted to show the -1 subsite lactone residue in an E3 or B3,O conformation [20]. On the basis of these early structures in combination with modeling, HEWL was proposed to bind its substrate in a distorted conformation in which the NAM residue binding in the -1 subsite adopted a boat-like conformation [20]. A product complex of chitopentaoside spanning the negative subsites up to -1, determined at low temperature, revealed the sugar residue in the -1 subsite to be in an undistorted 4C1 chair conformation [19]. More recently, complexes of HEWL with the modified substrate N-acetylglucosaminyl-(1,4)-2-deoxy-2-fluoroglycosyl fluoride and the HEWL E35Q mutant revealed a covalent bond to the nucleophile Glu35, with the -1 subsite sugar in a 4C1 chair conformation [9]. These structural data were collectively interpreted in light of kinetic studies to propose an electrophilic migration mechanism for HEWL in which the enzyme uses a 1,4BE34C1, or closely related, conformational itinerary [9].

The structure of α-lactalbumin is essentially identical in three-dimensional fold to HEWL. The first α-lactalbumin to have a high resolution structure determined was that from baboon [21]. Baboon lactalbumin shares conserved disulfide bonds and a large cleft equivalent to the substrate binding cleft in HEWL. α-Lactalbumins possess a conserved Ca2+ binding subsite, which in the baboon enzyme is a distorted pentagonal bipyramid comprised of Lys79, Asp82, Asp84, Asp87 and Asp88 and two water molecules. X-ray structures of the lactose synthase complex of murine α-lactalbumin bound to bovine galactosyltransferase have been determined [22]. The interface is comprised mainly of hydrophobic interactions. Binding of α-lactalbumin results in a large conformational change in galactosyltransferase that modifies the sugar nucleotide binding region conferring lactose synthase activity.

The lysozyme fold is shared by family GH19 chitinases, GH23 lysozymes, GH124 cellulases, and GH134 mannanases.

Family Firsts

First stereochemistry determination
Retention of glycosyl transfer to N-acetylglucosamine and methanol [23]. Quantitation of the transglycosylation to methanol of cleavage of radiolabelled disaccharides by HEWL showed reaction occurs with >99.7% retention of configuration [24].
First catalytic nucleophile identification
Asp52 of hen egg white lysozyme (HEWL) first proposed as stablizing an ion-pair intermediate [13]. Asp52 identified as nucleophile by X-ray crystallography of covalent complex formed with a 2-fluorosugar [9].
First general acid/base residue identification
Glu35 of HEWL proposed on the basis of X-ray structure of a complex with a chitooligosaccharide [13]; the HEWL Glu35Gln mutant displayed a loss of activity against bacterial cell wall [14].
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 [8].


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