Information on EC 1.14.99.53 - lytic chitin monooxygenase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
1.14.99.53
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RECOMMENDED NAME
GeneOntology No.
lytic chitin monooxygenase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(1->4)-N-acetyl-beta-D-glucosaminyl]n+m + reduced acceptor + O2 = [(1->4)-N-acetyl-beta-D-glucosaminyl]m-1-(1->4)-N-acetyl-2-deoxy-2-amino-D-glucono-1,5-lactone + [(1->4)-N-acetyl-beta-D-glucosaminyl]n + acceptor + H2O
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
chitin degradation III (Serratia)
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SYSTEMATIC NAME
IUBMB Comments
chitin, hydrogen-donor:oxygen oxidoreductase (N-acetyl-beta-D-glucosaminyl C1-hydroxylating/C4-dehdrogenating)
The enzyme cleaves chitin in an oxidative manner, releasing fragments of chitin with an N-acetylamino-D-glucono-1,5-lactone at the reducing end. The initially formed lactone at the reducing end of the shortened chitin chain quickly hydrolyses spontaneously to the aldonic acid. In vitro ascorbate can serve as reducing agent. The enzyme contains copper at the active site.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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UniProt
Manually annotated by BRENDA team
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E1UUV3
UniProt
Manually annotated by BRENDA team
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E1UUV3
UniProt
Manually annotated by BRENDA team
subsp. Kurstaki
UniProt
Manually annotated by BRENDA team
subsp. Kurstaki
UniProt
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
alpha-chitin + acceptor + O2
oligosaccharide aldonic acids + reduced acceptor + H2O
show the reaction diagram
alpha-chitin + ascorbate + O2
? + dehydroascorbate + H2O
show the reaction diagram
alpha-chitin + ascorbate + O2
C1-oxidized chitooligosaccharides + dehydroascorbate + H2O
show the reaction diagram
alpha-chitin + ascorbate + O2
chito-oligosaccharide aldonic acid + dehydroascorbate + H2O
show the reaction diagram
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products show a degree of polymerization from DP3 to DP8, with DP6 being the most abundant product after 24 h of incubation
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?
alpha-chitin + ascorbate + O2
oxidized chitooligosaccharides + dehydroascorbate + H2O
show the reaction diagram
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-
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?
alpha-chitin + ascorbate + O2
oxidized oligosaccharides + dehydroascorbate + H2O
show the reaction diagram
beta-chitin + acceptor + O2
chito-oligosaccharide aldonic acid + dehydroascorbate + H2O
show the reaction diagram
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products show a degree of polymerization from DP3 to DP8, with DP6 being the most abundant product after 24 h of incubation
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?
beta-chitin + acceptor + O2
oligosaccharide aldonic acids + reduced acceptor + H2O
show the reaction diagram
beta-chitin + ascorbate + O2
? + dehydroascorbate + H2O
show the reaction diagram
beta-chitin + ascorbate + O2
C1-oxidized chitooligosaccharides + dehydroascorbate + H2O
show the reaction diagram
beta-chitin + ascorbate + O2
C1-oxidized oligosaccharides + dehydroascorbate + H2O
show the reaction diagram
beta-chitin + ascorbate + O2
oxidized oligosaccharides + dehydroascorbate + H2O
show the reaction diagram
beta-chitin + ascorbic acid + O2
? + dehydroascorbate + H2O
show the reaction diagram
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-
-
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?
beta-chitin + cellobiose dehydrogenase + O2
C1-oxidized oligosaccharides + reduced cellobiose dehydrogenase + H2O
show the reaction diagram
chitin + acceptor + O2
? + reduced acceptor + H2O
show the reaction diagram
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?
chitin + acceptor + O2
C1-oxidized chito-oligosaccharides + reduced acceptor + H2O
show the reaction diagram
chitin + acceptor + O2
[chitin oligosaccharide]-N-acetyl-D-glucosaminate + [chitin oligosaccharide]-N-acetyl-D-glucosamino-1,5-lactone
show the reaction diagram
chitin + ascorbic acid + O2
? + dehydroascorbate + H2O
show the reaction diagram
colloidal chitin + ascorbate + O2
? + dehydroascorbate + H2O
show the reaction diagram
colloidal chitin + ascorbate + O2
[chitin oligosaccharide]-N-acetyl-D-glucosaminate + [chitin oligosaccharide]-N-acetyl-D-glucosamino-1,5-lactone + dehydroascorbate + H2O
show the reaction diagram
colloidal chitin chitin + ascorbate + O2
oxidized chitin oligosaccharides + dehydroascorbate + H2O
show the reaction diagram
crystalline chitin + ascorbate + O2
oxidized oligosaccharides + dehydroascorbate + H2O
show the reaction diagram
phosphoric acid swollen cellulase + ascorbic acid + O2
? + dehydroascorbate + H2O
show the reaction diagram
phosphoric acid swollen cellulose + ascorbate + O2
C4-oxidized cellooligosaccharides + C1/C4-oxidized cellooligosaccharides + dehydroascorbate + H2O
show the reaction diagram
reaction of EC 1.14.99.54
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?
[(1->4)-N-acetyl-beta-D-glucosaminyl]6 + ascorbate + O2
[(1->4)-N-acetyl-beta-D-glucosaminyl]3-(1->4)-N-acetyl-2-deoxy-2-amino-D-glucono-1,5-lactone + [(1->4)-N-acetyl-beta-D-glucosaminyl]2 + dehydroascorbate + H2O
show the reaction diagram
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[(1->4)-N-acetyl-beta-D-glucosaminyl]n+m + reduced acceptor + O2
[(1->4)-N-acetyl-beta-D-glucosaminyl]m-1-(1->4)-N-acetyl-2-deoxy-2-amino-D-glucono-1,5-lactone + [(1->4)-N-acetyl-beta-D-glucosaminyl]n + acceptor + H2O
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
copper
Zn2+
E1UUV3
may substitue for Cu2+. KD value 0.0081 mM at pH 5
additional information
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residues His28 and His114 in the catalytic center bind a variety of divalent metal ions such as Ca2+, Mg2+, Fe3+, Co2+, Zn2+, or Cu2+ with a clear preference for Cu2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cyanide
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mimic of molecular oxygen, binds to the metal ion only
NaCl
enzyme activity is decreased by NaCl
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
ammonium acetate buffer, 34% of maximum activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.8
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calculated
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
21000
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x * 21000, SDS-PAGE
24150
calculated from amino acid sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
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N-linked glycosylation
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in presence of Zn2+, to 1.55 A resolution, and in presence of Cu2+, to 1.4 AS resolution
analysis of the copper active site
E1UUV3
comparative analysis of sequences, solved structures, and homology models from AA9 and AA10 LPMO families.The two LPMO families are highly conserved, structurally they have minimal sequence similarity outside the active site residues
to 1.85 A resolution, tri-modular enzyme containing a catalytic family AA10 LPMO module, a family 5 chitin-binding module, and a C-terminal unclassified module which displays tight and specific binding to chitin
; crystal structure in the Cu(II)-bound form and photoreduction of the crystalline protein in the x-ray beam, leading to conversion from the initial Cu(II)-oxidized form with two coordinated water molecules, which adopts a trigonal bipyramidal geometry, to a reduced Cu(I) form in a T-shaped geometry with no coordinated water molecules
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comparative analysis of sequences, solved structures, and homology models from AA9 and AA10 LPMO families.The two LPMO families are highly conserved, structurally they have minimal sequence similarity outside the active site residues
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1.1 A resolution room-temperature X-ray structure and 2.1 A resolution neutron structure, show a putative dioxygen species equatorially bound to the active site copper with elongated density for the dioxygen, most consistent with a Cu(II)-bound peroxide
1.1 A resolution, room-temperature X-ray structure and a 2.1 A resolution neutron structure show a putative dioxygen species equatorially bound to the active site copper. Both structures show an elongated density for the dioxygen, consistent with a Cu(II)-bound peroxide. The coordination environment is consistent with Cu(II). The N-terminal amino group, involved in copper coordination, is present as a mixed neutral and deprotonated form
1.55 A resolution structure of N-terminal LPMO10A module reveals deletions of interacting loops that protrude from the core beta-sandwich scaffold in larger LPMO10s
crystallization at pH 3.5. Structure shows shows significant disorder of the active site in the absence of substrate ligand
structure of the catalytic domain, residues 37-230, to 1.08 A resolution. The active site in is formed by residues His-37 and His-144 that coordinate the copper atom in a T-shaped geometry
structures in the resting state and of a copper(II)-dioxo intermediate complex formed in the absence of substrate reveal pre-bound molecular oxygen adjacent to the active site. A conserved histidine is involved in promoting oxygen activation
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to 1.2 A resolution. Diffraction resolution and crystal morphology are improved by expression from a glycoengineered strain of Pichia pastoris
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calculation of solution structure. Ca2+, Mg2+, Fe3+, Co2+, Zn2+, or Cu2+ ions show binding to an interaction site located between His28 and His114
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molecular dynamics interactions between the LPMO and three different surface topologies of crystalline chitin. Most enzyme-substrate interactions involve the polysaccharide chain that is to be cleaved. Enzyme displays a constrained active site geometry as well as a tunnel connecting the bulk solvent to the copper site, through which only small molecules such as H2O, O2, and H2O2 can diffuse. Rearrangement of Cu-coordinating water molecules is necessary when binding the substrate and also provide a rationale for the experimentally observed C1 oxidative regiospecificity
structure of the catalytic domain, residues 37-230, to 1.08 A resolution. The active site in is formed by residues His-37 and His-144 that coordinate the copper atom in a T-shaped geometry
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to 1.3 A resolution
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
68.7
E1UUV3
melting temperature, presence of both copper and chitin
additional information
E1UUV3
chitin binding increases the thermal stability of the enzyme by 8.3 degrees, copper binding increases melting temperature by 21.6 degrees. Addition of chitin to the copper-bound enzyme increases thermal stability by additional 3.5 degrees
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression Bacillus subtilis yields a simple purification process without complicated periplasmic fractionation, as well as improved productivity
improved expression and purification protocol using Champion pET-SUMO vector, yielding high purity AA10 with yields in excess of 9 mg of protein per litre of culture
E1UUV3
simple purification method by affinity adsorption to obtain functional lytic polysaccharide monooxygenases. Purification follows a binding-elution protocol with low-grade polysaccharides including Avicel
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
codon-optimized expression in Escherichia coli
expressed in Escherichia coli BL21
expression in Baciluus subtilis
expression in Escherichia coli
expression in Pichia pastoris
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expression using a Brevibacillus-based expression system
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D140A
mutant shows moderately reduced activity and essentially unchanged oxidative regioselectivity
N85F
mutation changes the C1:C4 oxidation ratio from 0.9 (for the wild-type) to 5.9
W82Y
mutation changes the C1:C4 oxidation ratio from 0.9 (for the wild-type) to 2.0
W82Y/N85F
mutation changes the C1:C4 oxidation ratio from 0.9 (for the wild-type) to 10.9
W82Y/N85F/Q141W
mutation changes the C1:C4 oxidation ratio from 0.9 (for the wild-type) to 5.1
W82Y/N85F/Y116F
mutation changes the C1:C4 oxidation ratio from 0.9 (for the wild-type) to 14.7
W82Y/N85F/Y116F/Q141W
mutation changes the C1:C4 oxidation ratio from 0.9 (for the wild-type) to 5.8
Y116F
no changes in the C1:C4 oxidation ratio
Y54A
mutation of residue at subsite -4, minimal effect on degradation of beta-chitin, about 20% residual activity with substrate [(1->4)-N-acetyl-beta-D-glucosaminyl]6
A148G
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mutation leads to loss of C4 oxidation, i.e to the activity of EC 1.14.99.54
A148S
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mutation leads to loss of C4 oxidation, i.e to the activity of EC 1.14.99.54
W88Y/N91F
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mutation leads to loss of C4 oxidation, i.e to the activity of EC 1.14.99.54
A148G
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mutation leads to loss of C4 oxidation, i.e to the activity of EC 1.14.99.54
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A148S
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mutation leads to loss of C4 oxidation, i.e to the activity of EC 1.14.99.54
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additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
degradation
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presence of lytic polysaccharide monooxygenase CBP21 facilitates the degradation of chitin substrates (colloidal chitin, beta-chitin, and alpha-chitin) by Chi92
synthesis