DB code: T00063

RLCP classification	1.32.5000.73 : Hydrolysis
CATH domain	2.60.40.10 : Immunoglobulin-like
	3.20.20.80 : TIM Barrel	Catalytic domain
	3.10.50.10 : Chitinase A; domain 3
E.C.	3.2.1.14
CSA	1ctn
M-CSA	1ctn
MACiE

CATH domain	Related DB codes (homologues)
3.10.50.10 : Chitinase A; domain 3	M00134
2.60.40.10 : Immunoglobulin-like	M00131 T00257 T00005 M00113 M00127 M00132 M00323 M00325 M00327 M00329 M00330 M00331 M00332 T00307 D00166 D00500 M00112 M00193 T00065 T00067 T00245
3.20.20.80 : TIM Barrel	S00202 S00210 S00748 S00906 S00907 S00911 S00912 S00915 M00134 M00160 D00479 S00204 S00205 S00206 S00207 S00203 S00208 S00209 S00211 S00213 S00214 M00113 T00307 D00165 D00166 D00169 D00176 D00501 D00502 D00503 D00844 D00861 D00864 M00026 M00112 M00193 M00346 T00057 T00062 T00066 T00067

Uniprot Enzyme Name
UniprotKB	Protein name	Synonyms	CAZy	Pfam
P07254	Chitinase A	EC 3.2.1.14	GH18 (Glycoside Hydrolase Family 18)	PF08329 (ChitinaseA_N) PF00704 (Glyco_hydro_18) [Graphical View]

KEGG enzyme name
chitinase chitodextrinase 1,4-beta-poly-N-acetylglucosaminidase poly-beta-glucosaminidase beta-1,4-poly-N-acetyl glucosamidinase poly[1,4-(N-acetyl-beta-D-glucosaminide)] glycanohydrolase

UniprotKB: Accession Number	Entry name	Activity	Subunit	Subcellular location	Cofactor
P07254	CHIA_SERMA	Random hydrolysis of N-acetyl-beta-D- glucosaminide (1->4)-beta-linkages in chitin and chitodextrins.

KEGG Pathways
Map code	Pathways	E.C.
MAP00530	Aminosugars metabolism

Compound table
	Substrates			Products			Intermediates
KEGG-id	C00461	C00851	C00001	C03518	C00140	C00461
E.C.
Compound	Chitin	Chitodextrin	H2O	N-Acetyl-D-glucosaminide	N-Acetyl-D-glucosamine	Chitin
Type	amide group,polysaccharide	amide group,polysaccharide	H2O	amide group,carbohydrate	amide group,carbohydrate	amide group,polysaccharide
ChEBI			15377 15377		506227 506227
PubChem			22247451 962 22247451 962		439174 439174

1ctnA01	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound
1edqA01	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound
1ehnA01	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound
1eibA01	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound
1ffrA01	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound
1k9tA01	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound
1nh6A01	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound
1ctnA02	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound
1edqA02	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound
1ehnA02	Bound:NAG-NAG-NAG-NAG-NAG-NAG-NAG-NAG	Unbound		Unbound	Unbound	Unbound	Unbound
1eibA02	Bound:NAG-NAG-NAG-NAG-NAG-NAG-NAG-NAG	Unbound		Unbound	Unbound	Unbound	Unbound
1ffrA02	Unbound	Unbound		Unbound	Unbound	Unbound	Transition-state-bound:NAG-NAG-NAG-NAG-NAG (chain B), NAG-NAG (chain C)
1k9tA02	Bound:NAG-NAG-NAG-NAG	Unbound		Unbound	Unbound	Unbound	Unbound
1nh6A02	Bound:NAG-NAG-NAG-NAG-NAG-NAG	Unbound		Unbound	Unbound	Unbound	Unbound
1ctnA03	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound
1edqA03	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound
1ehnA03	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound
1eibA03	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound
1ffrA03	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound
1k9tA03	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound
1nh6A03	Unbound	Unbound		Unbound	Unbound	Unbound	Unbound

Reference for Active-site residues
resource	references	E.C.
literature [6]

Active-site residues
PDB	Catalytic residues	Cofactor-binding residues	Modified residues	Main-chain involved in catalysis	Comment

1ctnA01
1edqA01
1ehnA01
1eibA01
1ffrA01
1k9tA01
1nh6A01
1ctnA02	ASP 313;GLU 315;TYR 390;ASP 391
1edqA02	ASP 313;GLU 315;TYR 390;ASP 391
1ehnA02	ASP 313;;TYR 390;ASP 391				mutant E315Q
1eibA02	;GLU 315;TYR 390;ASP 391				mutant D313A
1ffrA02	ASP 313;GLU 315;;ASP 391				mutant Y390F
1k9tA02	ASP 313;GLU 315;TYR 390;				mutant D391A
1nh6A02	ASP 313;;TYR 390;ASP 391				mutant E315L
1ctnA03
1edqA03
1ehnA03
1eibA03
1ffrA03
1k9tA03
1nh6A03

References for Catalytic Mechanism
References	Sections	No. of steps in catalysis
[4]	p.202-203
[6]	Scheme 1, p.11341-11343	2
[8]	p.403

References
[1]
Resource
Comments	REVISIONS, AND X-RAY CRYSTALLOGRAPHY (2.3 ANGSTROMS).
Medline ID	95219379
PubMed ID	7704527
Journal	Structure
Year	1994
Volume	2
Pages	1169-80
Authors	Perrakis A, Tews I, Dauter Z, Oppenheim AB, Chet I, Wilson KS, Vorgias CE
Title	Crystal structure of a bacterial chitinase at 2.3 A resolution.
Related PDB	1ctn
Related UniProtKB	P07254
[2]
Resource
Comments
Medline ID
PubMed ID	8831791
Journal	J Mol Biol
Year	1996
Volume	262
Pages	243-57
Authors	Terwisscha van Scheltinga AC, Hennig M, Dijkstra BW
Title	The 1.8 A resolution structure of hevamine, a plant chitinase/lysozyme, and analysis of the conserved sequence and structure motifs of glycosyl hydrolase family 18.
Related PDB
Related UniProtKB
[3]
Resource
Comments
Medline ID
PubMed ID	9377712
Journal	Fold Des
Year	1997
Volume	2
Pages	291-4
Authors	Perrakis A, Ouzounis C, Wilson KS
Title	Evolution of immunoglobulin-like modules in chitinases: their structural flexibility and functional implications.
Related PDB
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID	10794597
Journal	IUBMB Life
Year	1999
Volume	48
Pages	199-204
Authors	Lin FP, Chen HC, Lin CS
Title	Site-directed mutagenesis of Asp313, Glu315, and Asp391 residues in chitinase of Aeromonas caviae.
Related PDB
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID	10823940
Journal	Proc Natl Acad Sci U S A
Year	2000
Volume	97
Pages	5842-7
Authors	van Aalten DM, Synstad B, Brurberg MB, Hough E, Riise BW, Eijsink VG, Wierenga RK
Title	Structure of a two-domain chitotriosidase from Serratia marcescens at 1.9-A resolution.
Related PDB
Related UniProtKB
[6]
Resource
Comments	X-ray crystallography
Medline ID
PubMed ID	11560481
Journal	Biochemistry
Year	2001
Volume	40
Pages	11338-43
Authors	Papanikolau Y, Prag G, Tavlas G, Vorgias CE, Oppenheim AB, Petratos K
Title	High resolution structural analyses of mutant chitinase A complexes with substrates provide new insight into the mechanism of catalysis.
Related PDB	1ehn 1eib 1ffr
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID	11342059
Journal	Biochim Biophys Acta
Year	2001
Volume	1545
Pages	349-56
Authors	Lonhienne T, Baise E, Feller G, Bouriotis V, Gerday C
Title	Enzyme activity determination on macromolecular substrates by isothermal titration calorimetry: application to mesophilic and psychrophilic chitinases.
Related PDB
Related UniProtKB
[8]
Resource
Comments	X-ray crystallography
Medline ID
PubMed ID	12554965
Journal	Acta Crystallogr D Biol Crystallogr
Year	2003
Volume	59
Pages	400-3
Authors	Papanikolau Y, Tavlas G, Vorgias CE, Petratos K
Title	De novo purification scheme and crystallization conditions yield high-resolution structures of chitinase A and its complex with the inhibitor allosamidin.
Related PDB	1edq
Related UniProtKB
[9]
Resource
Comments
Medline ID
PubMed ID	12932195
Journal	Biochem J
Year	2003
Volume	376
Pages	87-95
Authors	Aronson NN Jr, Halloran BA, Alexyev MF, Amable L, Madura JD, Pasupulati L, Worth C, Van Roey P
Title	Family 18 chitinase-oligosaccharide substrate interaction: subsite preference and anomer selectivity of Serratia marcescens chitinase A.
Related PDB	1nh6
Related UniProtKB

Comments
(5) The deprotonated sidechain of Glu315 acts as a general base, abstracting the water molecule, which can act on the C1 carbon at subsite (-1). Taken together, Glu315 acts as an acid-base, whilst Asp313 modulates the role of Glu315. Moreover, Tyr390 stabilizes the transition-state through the water. The stabilization might be assisted by N-acetyl group of the substrate. This enzyme is composed of the N-terminal immunoglobulin-like region and the C-terminal catalytic region, which has also two distinct domains. According to the literature [3], the N-terminal immunoglobulin-like domain might interact with the chitin substrate during catalysis. In the early study [4], Asp313, Glu315 and Asp391 have been thought to be involved in catalysis. The paper [4] suggested that Asp313 and Glu315 might act as a stabilizer and a general acid, respectively. According to the more recent work ([6] & [8]), however, Tyr390 was reported to be involved in the catalytic reaction, instead of Asp391. The paper [6] proposed the following mechanism: (1) The sugar unit at the subsite (-1) adopts a boat conformation, which has higher free energy by 8kcal/mol, and its N-acetyl group points toward Asp313 and Glu315. The side chain of Asp313 points toward Asp311, away from Glu315. A water molecule is hydrogen-bonded to the phenol hydroxyl group of Tyr390 and the amine group of the acetamido group of the sugar unit at the subsite (-1). The sidechain carboxylate of Glu315 is protonated. (2) Glu315 functions as a general acid, and protonates the O4 oxygen of the sugar unit at the subsite (+1), thus cleaving the glycosidic linkage, O4(+1)-C1(-1). (This suggests SN1-like cleavage.) (3) The positive charge is developed on C1 and O5 atoms at the subsite (-1), which can be stabilized by the water molecule hydrogen-bonded to Tyr390 and the acetamido group at (-1), the O7 atom of the acetamido group at (-1), and the deprotonated sidechain of Glu315. (4) This cleavage induces the rotation of Asp313 toward Glu315, and the rotation of the acetamido group at (-1) toward Tyr390. The rotation of the acetamido group translocates the water molecule bound to Tyr390 closer to Glu315. However, the mutation of Asp391 inactivates this enzyme, suggesting that the residue must be involved in catalysis (see Table 1 of [6]).

Comments

(5) The deprotonated sidechain of Glu315 acts as a general base, abstracting the water molecule, which can act on the C1 carbon at subsite (-1).
Taken together, Glu315 acts as an acid-base, whilst Asp313 modulates the role of Glu315. Moreover, Tyr390 stabilizes the transition-state through the water. The stabilization might be assisted by N-acetyl group of the substrate.
This enzyme is composed of the N-terminal immunoglobulin-like region and the C-terminal catalytic region, which has also two distinct domains. According to the literature [3], the N-terminal immunoglobulin-like domain might interact with the chitin substrate during catalysis.
In the early study [4], Asp313, Glu315 and Asp391 have been thought to be involved in catalysis. The paper [4] suggested that Asp313 and Glu315 might act as a stabilizer and a general acid, respectively.
According to the more recent work ([6] & [8]), however, Tyr390 was reported to be involved in the catalytic reaction, instead of Asp391. The paper [6] proposed the following mechanism:
(1) The sugar unit at the subsite (-1) adopts a boat conformation, which has higher free energy by 8kcal/mol, and its N-acetyl group points toward Asp313 and Glu315. The side chain of Asp313 points toward Asp311, away from Glu315. A water molecule is hydrogen-bonded to the phenol hydroxyl group of Tyr390 and the amine group of the acetamido group of the sugar unit at the subsite (-1). The sidechain carboxylate of Glu315 is protonated.
(2) Glu315 functions as a general acid, and protonates the O4 oxygen of the sugar unit at the subsite (+1), thus cleaving the glycosidic linkage, O4(+1)-C1(-1). (This suggests SN1-like cleavage.)
(3) The positive charge is developed on C1 and O5 atoms at the subsite (-1), which can be stabilized by the water molecule hydrogen-bonded to Tyr390 and the acetamido group at (-1), the O7 atom of the acetamido group at (-1), and the deprotonated sidechain of Glu315.
(4) This cleavage induces the rotation of Asp313 toward Glu315, and the rotation of the acetamido group at (-1) toward Tyr390. The rotation of the acetamido group translocates the water molecule bound to Tyr390 closer to Glu315.
However, the mutation of Asp391 inactivates this enzyme, suggesting that the residue must be involved in catalysis (see Table 1 of [6]).

Created	Updated
2004-04-30	2009-02-26