DB code: S00341

RLCP classification	1.13.30100.49 : Hydrolysis
CATH domain	3.40.50.850 : Rossmann fold	Catalytic domain
E.C.	3.5.1.59
CSA	1nba
M-CSA	1nba
MACiE	M0025

CATH domain	Related DB codes (homologues)

Uniprot Enzyme Name
UniprotKB	Protein name	Synonyms	Pfam
P32400	N-carbamoylsarcosine amidase	EC 3.5.1.59 N-carbamoylsarcosine amidohydrolase CSHase	PF00857 (Isochorismatase) [Graphical View]

KEGG enzyme name
N-carbamoylsarcosine amidase carbamoylsarcosine amidase

UniprotKB: Accession Number	Entry name	Activity	Subunit	Subcellular location	Cofactor
P32400	CSH_ARTSP	N-carbamoylsarcosine + H(2)O = sarcosine + CO(2) + NH(3).	Homotetramer.		Binds 1 sulfate ion per subunit.

KEGG Pathways
Map code	Pathways	E.C.
MAP00330	Arginine and proline metabolism

Compound table
	Cofactors	Substrates		Products			Intermediates
KEGG-id	C00059	C01043	C00001	C00213	C00011	C00014
E.C.
Compound	Sulfate	N-Carbamoylsarcosine	H2O	Sarcosine	CO2	NH3
Type	sulfate group	amino acids,amide group,amine group	H2O	amino acids	others	amine group,organic ion
ChEBI	26836 26836	15737 15737	15377 15377	15611 57433 15611 57433	16526 16526	16134 16134
PubChem	1118 22066174 5152822 1118 22066174 5152822	439375 439375	22247451 962 22247451 962	1088 7311726 1088 7311726	280 280	222 222

1nbaA	Bound:SO4	Unbound		Unbound	Unbound	Unbound
1nbaB	Bound:SO4	Unbound		Unbound	Unbound	Unbound
1nbaC	Bound:SO4	Unbound		Unbound	Unbound	Unbound
1nbaD	Bound:SO4	Unbound		Unbound	Unbound	Unbound

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

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

1nbaA	ALA 172;THR 173(cis-peptide);ASP 51;LYS 144;CYS 177
1nbaB	ALA 172;THR 173(cis-peptide);ASP 51;LYS 144;CYS 177
1nbaC	ALA 172;THR 173(cis-peptide);ASP 51;LYS 144;CYS 177
1nbaD	ALA 172;THR 173(cis-peptide);ASP 51;LYS 144;CYS 177

References for Catalytic Mechanism
References	Sections	No. of steps in catalysis
[1]	p.1128
[2]	p.278-279
[3]	p.256-257	8
[4]	Fig.6	5

References
[1]
Resource
Comments	X-ray crystallography (2.0 Angstroms)
Medline ID
PubMed ID	1381445
Journal	J Mol Biol
Year	1992
Volume	226
Pages	1111-30
Authors	Romao MJ, Turk D, Gomis-Ruth FX, Huber R, Schumacher G, Mollering H, Russmann L
Title	Crystal structure analysis, refinement and enzymatic reaction mechanism of N-carbamoylsarcosine amidohydrolase from Arthrobacter sp. at 2.0 A resolution.
Related PDB	1nba
Related UniProtKB
[2]
Resource
Comments	X-ray crystallography (2.28 Angstroms)
Medline ID	97070380
PubMed ID	8913306
Journal	J Mol Biol
Year	1996
Volume	263
Pages	269-83
Authors	Zajc A., Romao M.J., Turk D., Huber R
Title	Crystallographic and fluorescence studies of ligand binding to N-carbamoylsarcosine amidohydrolase from Arthrobacter sp.
Related PDB
Related UniProtKB
[3]
Resource
Comments
Medline ID
PubMed ID	11237598
Journal	J Mol Biol
Year	2001
Volume	306
Pages	251-61
Authors	Wang WC, Hsu WH, Chien FT, Chen CY
Title	Crystal structure and site-directed mutagenesis studies of N-carbamoyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter reveals a homotetramer and insight into a catalytic cleft.
Related PDB
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID	11714269
Journal	Biochemistry
Year	2001
Volume	40
Pages	14166-72
Authors	Du X, Wang W, Kim R, Yakota H, Nguyen H, Kim SH
Title	Crystal structure and mechanism of catalysis of a pyrazinamidase from Pyrococcus horikoshii.
Related PDB
Related UniProtKB

Comments
All the literature reported that Cys177 of this enzyme, CSHase, is the nucleophilic residue that will attack the carbamoyl carbon atom of the substrate to form a covalent bond with the atom (see [1], [2], [3] & [4]). The paper on the other enzyme, which is not homologous but has got a similar active site, and then probably will adopt the similar catalytic mechanism, mentioned that CSHase also have the Cys-Asp-Lys catalytic triad. According to the literature [4] on the homologous enzyme of CSHase, Asp51, Lys144 and Cys177 form the catalytic triad, whilst the consecutive cis-peptide bond residues, Ala172-Thr173, could play an important role in stablizing the intermediate by forming the oxyanion hole, which is observed in the structures of serine proteases. The paper [3] proposed a plausible catalytic mechanism that consists of two stages, as follows; The first stage is an acylation reaction of CSHase with the carbamoyl moiety, resulting in a cleavage of an NH3 molecule; (1) the adjacent carboxyl group of Asp51 abstracts the proton of the -SH group of Cys177, (2) the sulfur atom of Cys177 carries out a nucleophilic attack on the carbon atom of the carbonyl group, (3) the NZ atom of Lys144 possibly stabilizes an acquired transient tetrahedral intermediate carrying an oxyanion near it, (4) the proton held by Asp51 is then donated to the nitrogen atom of the susceptible C-N bond, which thus is cleaved and produces an ammonia molecule. The second stage involves the deacylation of the acylated enzyme intermediate. A water molecule might take the place occupied ealier by the amine component of the substrate, followed by reactions similar to those at the first stage, with water substituting for the amine component: (5) Asp51 draws a proton away from water, (6) the resulting OH- ion attacks the carbonyl carbon atom of the acyl group that is attached to Cys177, (7) a transient tetrahedral intermediate is formed, which is stabilized by Lys144, and (8) Asp51 then donates the proton to the sulfur atom of Cys177, which then releases the acid component that may further decomposes into a sarcosine and CO2 molecule. Moreover, although the sulfate is annotated as cofactor, it does not seem to be directly involved in catalysis.

Comments

All the literature reported that Cys177 of this enzyme, CSHase, is the nucleophilic residue that will attack the carbamoyl carbon atom of the substrate to form a covalent bond with the atom (see [1], [2], [3] & [4]).
The paper on the other enzyme, which is not homologous but has got a similar active site, and then probably will adopt the similar catalytic mechanism, mentioned that CSHase also have the Cys-Asp-Lys catalytic triad. According to the literature [4] on the homologous enzyme of CSHase, Asp51, Lys144 and Cys177 form the catalytic triad, whilst the consecutive cis-peptide bond residues, Ala172-Thr173, could play an important role in stablizing the intermediate by forming the oxyanion hole, which is observed in the structures of serine proteases.
The paper [3] proposed a plausible catalytic mechanism that consists of two stages, as follows;
The first stage is an acylation reaction of CSHase with the carbamoyl moiety, resulting in a cleavage of an NH3 molecule;
(1) the adjacent carboxyl group of Asp51 abstracts the proton of the -SH group of Cys177,
(2) the sulfur atom of Cys177 carries out a nucleophilic attack on the carbon atom of the carbonyl group,
(3) the NZ atom of Lys144 possibly stabilizes an acquired transient tetrahedral intermediate carrying an oxyanion near it,
(4) the proton held by Asp51 is then donated to the nitrogen atom of the susceptible C-N bond, which thus is cleaved and produces an ammonia molecule.
The second stage involves the deacylation of the acylated enzyme intermediate. A water molecule might take the place occupied ealier by the amine component of the substrate, followed by reactions similar to those at the first stage, with water substituting for the amine component:
(5) Asp51 draws a proton away from water,
(6) the resulting OH- ion attacks the carbonyl carbon atom of the acyl group that is attached to Cys177,
(7) a transient tetrahedral intermediate is formed, which is stabilized by Lys144, and
(8) Asp51 then donates the proton to the sulfur atom of Cys177, which then releases the acid component that may further decomposes into a sarcosine and CO2 molecule.
Moreover, although the sulfate is annotated as cofactor, it does not seem to be directly involved in catalysis.

Created	Updated
2002-07-09	2009-02-26