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Difference between revisions of "Glycoside Hydrolase Family 22"

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[[Glycoside hydrolase]] family 22 contains proteins with two main functions: lysozymes and α-lactalbumin.
 
[[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.
+
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 <cite>Dobson1984 Mackie2002</cite>. Defects in the gene encoding human lysozyme can result in a rare hereditary condition, amyloidosis VIII, in which lysozyme deposits as a protein aggregate <cite>Jeyashekar2005</cite>.
  
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.
+
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 <cite>Prager1988 Qasba1997</cite>. α-Lactalbumins possess a conserved Ca<sup>2+</sup> binding site, with a high affinity for the cation of 3-5 nM <cite>Mitani1986</cite>. α-Lactalbumin possesses no meaningful catalytic activity against peptidoglycan, nor does it bind chitooligosaccharides.
  
 
== Kinetics and Mechanism ==
 
== Kinetics and Mechanism ==
HEWL operates 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 through the use of the substrate chitobiosyl fluoride and the HEWL E35Q (acid/base) mutant, detected by mass spectrometry; use of the modified substrate ''N''-acetylglucosaminyl-(1,4)-2-deoxy-2-fluoroglycosyl fluoride and the HEWL E35Q mutant, andd detected by mass spectrometry and studied by X-ray crystallography <cite>Vocadlo2001</cite>. 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 <cite>Dahlquist1969 Rand-Meir1969</cite>, and thus that the 2-acetamido functionality is not critical for catalysis. The HEWL-catalyzed hydrolysis of phenyl chitobioside afforded secondary kinetic isotope effects for H1/D1 of ''k''<sub>H</sub>/''k<sub>D</sub> 1.11, revealing considerable oxocarbenium ion character for the glycosylation transition state of the enzyme-catalyzed reaction <cite>Dahlquist1969</cite>.
+
HEWL was the first enzyme for which the 3-dimensional structure was solved <cite>Blake1962 Blake1965</cite>. 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 <cite>Vocadlo2001</cite>. 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 <cite>Dahlquist1969 Rand-Meir1969</cite>, 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 ''K''<sub>M</sub> values, but varying ''k''<sub>cat</sub> values, giving support for a rate-determining step involving concerted acid-base or acid-nucleophilic catalysis <cite>Lowe1967</cite>.  
 
 
α-Lactalbumins possess a conserved Ca<sup>2+</sup> binding site, with a high affinity for the cation of 3-5 nM <cite>Mitani1986</cite>.
 
  
 +
HEWL-catalyzed hydrolysis of phenyl chitobioside afforded secondary kinetic isotope effects for substrates isotopologous at the anomeric centre (H1/D1) of ''k''<sub>H</sub>/''k''<sub>D</sub> 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 <cite>Dahlquist1969</cite>. 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)<sub>3</sub>, or the chitin hexasaccharide (NAG)<sub>6</sub>, demonstrating that the enzyme possesses 6 subsites.
  
 
== Catalytic Residues ==
 
== Catalytic Residues ==
Inspection of complexes of lysozyme with chitooligosaccharides and chemical reasoning led to the proposal of Glu35 as a proton donor <cite>Blake1967</cite>. Site directed mutagenesis of Glu35 to Gln35 resulted in a complete loss of activity against ''Micrococcus luteus'' cell wall <cite>Malcolm1989</cite>. 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 <cite>Blake1967</cite>. The Asp52Asn mutant exhibited approximately 5% wild-type lytic ability against ''Micrococcus luteus'' cell wall <cite>Malcolm1989</cite>. 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 <cite>Vocadlo2001</cite>.
+
Inspection of complexes of lysozyme with chitooligosaccharides and chemical reasoning led to the proposal of Glu35 as a proton donor <cite>Blake1967</cite>. Site directed mutagenesis of Glu35 to Gln35 provided a mutant with no activity against ''Micrococcus luteus'' cell wall <cite>Malcolm1989</cite>. 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 <cite>Blake1967</cite>. An early example of unnatural amino acid mutagenesis realized by chemical mutagenesis of Asp52 to Homoser52 yielded an enzyme with greatly reduced catalytic activity <cite>Eshdat1974</cite>. Unexpectedly, the Asp52Asn mutant exhibited approximately 5% wild-type lytic ability against ''Micrococcus luteus'' cell wall <cite>Malcolm1989</cite> 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 <cite>Matsumura1996</cite>. 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 <cite>Vocadlo2001</cite>.
  
α-Lactalbumins typically lack the conserved catalytic residues present in lysozymes.
+
α-Lactalbumins typically lack the conserved catalytic residues present in lysozymes. Two naturally occurring variants of human lysozyme, Ile56Thr and Asp67His, are amyloidogenic <cite>Jeyashekar2005</cite>. 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 ==
 
== Three-dimensional structures ==
The first structure of a GH22 member was that of hen egg white lysozyme (HEWL) <cite>Blake1962 Blake1965</cite>. 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. 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. 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 runs across the face of the structure that comprises the active site and contains the catalytic residues Glu35 and Asp52. Four disulfide bonds are present in the structure: Cys6-Cys1127, Cys30-Cys115, Cys64-Cys80, and Cys76-Cys94.  
+
[http://www.cazy.org/GH22_structure.html 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 <cite>Blake1962 Blake1965</cite>. 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 <cite> Goodsell2000</cite>. 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.  
  
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 <cite>Acharya1989</cite>. Baboon lactalbumin shares conserved disulfide bonds and a large cleft equivalent to the substrate binding cleft in HEWL. α-Lactalbumin possesses a Ca<sup>2+</sup> binding subsite, which in the baboon enzyme is a distorted pentagonal bipyramid comprised of Lys79, Asp82, Asp84, Asp87 and Asp88 and two water molecules.
+
[http://www.cazy.org/GH22_structure.html 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 <cite>Strynadka1991</cite>; however, interpretation of this complex suffers from considerable disorder manifested as high temperature factors associated with the sugar bound in the -1 subsite <cite>Davies2009</cite>. 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 ''E''<sub>3</sub> or ''B''<sub>3,O</sub> conformation <cite>Ford1974</cite>. 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 <cite>Ford1974</cite>. 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 <sup>4</sup>''C''<sub>1</sub> chair conformation <cite>Davies2009</cite>. 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 <sup>4</sup>''C''<sub>1</sub> chair conformation <cite>Vocadlo2001</cite>. 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 <sup>1,4</sup>''B'' &rarr; ''E''<sub>3</sub> &rarr; <sup>4</sup>''C''<sub>1</sub>, or closely related, conformational itinerary <cite>Vocadlo2001</cite>.
 +
 
 +
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 <cite>Acharya1989</cite>. Baboon lactalbumin shares conserved disulfide bonds and a large cleft equivalent to the substrate binding cleft in HEWL. α-Lactalbumins possess a conserved Ca<sup>2+</sup> 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 <cite>Ramakrishnan2001</cite>. 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.
 
The lysozyme fold is shared by family [[GH19]] chitinases, [[GH23]] lysozymes, [[GH124]] cellulases, and [[GH134]] mannanases.
  
 
== Family Firsts ==
 
== Family Firsts ==
;First stereochemistry determination: Retention of glycosyl transfer to ''N''-acetylglucosamine and methanol <cite>Rupley1967</cite>.
+
;First stereochemistry determination: Retention of glycosyl transfer to ''N''-acetylglucosamine and methanol <cite>Rupley1967</cite>. Quantitation of the transglycosylation to methanol of cleavage of radiolabelled disaccharides by HEWL showed reaction occurs with >99.7% retention of configuration <cite>Dahlquist1969a</cite>.
;First catalytic nucleophile identification: Asp52 of hen egg white lysozyme (HEWL), by X-ray crystallography of covalent complex formed with a 2-fluorosugar <cite>Vocadlo2001</cite>.
+
;First catalytic nucleophile identification: Asp52 of hen egg white lysozyme (HEWL) first proposed as stablizing an ion-pair intermediate <cite>Blake1967</cite>. Asp52 identified as nucleophile by X-ray crystallography of covalent complex formed with a 2-fluorosugar <cite>Vocadlo2001</cite>.
 
;First general acid/base residue identification: Glu35 of HEWL proposed on the basis of X-ray structure of a complex with a chitooligosaccharide <cite>Blake1967</cite>; the HEWL Glu35Gln mutant displayed a loss of activity against bacterial cell wall <cite>Malcolm1989</cite>.
 
;First general acid/base residue identification: Glu35 of HEWL proposed on the basis of X-ray structure of a complex with a chitooligosaccharide <cite>Blake1967</cite>; the HEWL Glu35Gln mutant displayed a loss of activity against bacterial cell wall <cite>Malcolm1989</cite>.
 
;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 <cite>Blake1965</cite>.
 
;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 <cite>Blake1965</cite>.
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<biblio>
 
<biblio>
 
#Acharya1989 pmid=2769757
 
#Acharya1989 pmid=2769757
 
 
#Blake1962 pmid=13971463
 
#Blake1962 pmid=13971463
 
#Blake1965 pmid=5891407
 
#Blake1965 pmid=5891407
 
#Blake1967 pmid=4382801
 
#Blake1967 pmid=4382801
 +
#Dahlquist1969a pmid=5815782
 
#Dahlquist1969 pmid=5388150
 
#Dahlquist1969 pmid=5388150
 +
#Davies2009 Davies GJ, Withers SG, Vocadlo DJ. The Chitopentaose Complex of a Mutant Hen Egg-White Lysozyme Displays No Distortion of the −1 Sugar Away from a 4C1 Chair Conformation. Aust. J. Chem. 2009, 62, 528–532. [http://dx.doi.org/ 10.1071/CH09038]
 +
#Dobson1984 pmid=6432801
 +
#Eshdat1974 pmid=4525456
 +
#Ford1974 pmid=4453000
 +
#Goodsell2000 Goodsell DS, ''Lysozyme'', RCSB PDB Molecule of the Month, September 2000, [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_9 DOI:10.2210/rcsb_pdb/mom_2000_9]
 +
#Jeyashekar2005 pmid=15657495
 +
#Lowe1967 pmid=6049930
 
#Malcolm1989 pmid=2563161
 
#Malcolm1989 pmid=2563161
 +
#Mackie2002 pmid=21708724
 +
#Matsumura1996 pmid=8639670
 
#Mitani1986 pmid=3087980
 
#Mitani1986 pmid=3087980
 +
#Prager1988 pmid=3146643
 +
#Qasba1997 pmid=9307874
 +
#Ramakrishnan2001 pmid=11419947
 
#Rand-Meir1969 pmid=5346398
 
#Rand-Meir1969 pmid=5346398
 
#Rupley1967 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. [http://www.pnas.org/content/57/3/496.short] pmcid=PMC335536
 
#Rupley1967 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. [http://www.pnas.org/content/57/3/496.short] pmcid=PMC335536
 +
#Strynadka1991 pmid=1856865
 
#Vocadlo2001 pmid=11518970
 
#Vocadlo2001 pmid=11518970
 
</biblio>
 
</biblio>
  
 
[[Category:Glycoside Hydrolase Families|GH022]]
 
[[Category:Glycoside Hydrolase Families|GH022]]

Latest revision as of 13:19, 18 December 2021

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


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].

References

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