N-carbamoylsarcosine amidase

 

Carbamoylsarcosine amidohydrolase (CSHase) is involved in one of two alternative creatine degradative pathways in micro-organisms. Both pathways start with creatinine and end with glycine, but the CSHase containing pathway proceeds via N-methylhydantoin, N-carbamoylsarcosine, and sarcosine.

 

Reference Protein and Structure

Sequence
P32400 UniProt (3.5.1.59) IPR000868 (Sequence Homologues) (PDB Homologues)
Biological species
Arthrobacter sp. (Bacteria) Uniprot
PDB
1nba - CRYSTAL STRUCTURE ANALYSIS, REFINEMENT AND ENZYMATIC REACTION MECHANISM OF N-CARBAMOYLSARCOSINE AMIDOHYDROLASE FROM ARTHROBACTER SP. AT 2.0 ANGSTROMS RESOLUTION (2.0 Å) PDBe PDBsum 1nba
Catalytic CATH Domains
3.40.50.850 CATHdb (see all for 1nba)
Click To Show Structure

Enzyme Reaction (EC:3.5.1.59)

water
CHEBI:15377ChEBI
+
N-carbamoylsarcosine
CHEBI:15737ChEBI
→
carbon dioxide
CHEBI:16526ChEBI
+
ammonia
CHEBI:16134ChEBI
+
sarcosine
CHEBI:15611ChEBI
Alternative enzyme names: Carbamoylsarcosine amidase, CSHase,

Enzyme Mechanism

Introduction

Cys177 is activated as a nucleophile by the general base Asp51, attacking the carbamoyl group of the substrate. The resulting anionic tretrahedral intermediate is stabilised by the cationic lysine side chain of Lys144. Ammonia is eliminated from the enzyme-substrate adduct intermediate. Hydrolysis eliminates Cys177, producing sarcosine, CO2 and NH3. The release of ammonia is thought to be promoted through electrostatic interactions with Asp51.

Catalytic Residues Roles

UniProt PDB* (1nba)
Asp51 Asp51A Acts as a general acid/base, abstracting a proton from the Cys nucleophile and later the nucleophilic water molecule. In both cases it is returned to its initial protonation state by the leaving group. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Ala172 (main-C), Thr173 Ala172A (main-C), Thr173A Help stabilise the reactive intermediates. hydrogen bond acceptor, electrostatic stabiliser
Lys144 Lys144A Activates the nucleophilic Cys by altering its pKa. activator, hydrogen bond donor, electrostatic stabiliser
Cys177 Cys177A Acts as the catalytic nucleophile. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleophile, proton acceptor, proton donor, nucleofuge
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

bimolecular nucleophilic addition, overall reactant used, enzyme-substrate complex formation, intermediate formation, proton transfer, unimolecular elimination by the conjugate base, overall product formed, deamination, enzyme-substrate complex cleavage, intermediate collapse, intermediate terminated, native state of enzyme regenerated, reaction occurs outside the enzyme

References

  1. Nakai T et al. (2000), Structure, 8, 729-738. Crystal structure of N-carbamyl-d-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases. DOI:10.1016/s0969-2126(00)00160-x. PMID:10903946.
  2. Luo HB et al. (2010), J Struct Biol, 169, 304-311. Crystal structure and molecular modeling study of N-carbamoylsarcosine amidase Ta0454 from Thermoplasma acidophilum. DOI:10.1016/j.jsb.2009.11.008. PMID:19932181.
  3. Wang WC et al. (2001), J Mol Biol, 306, 251-261. 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 cleft11Edited by R. Huber. DOI:10.1006/jmbi.2000.4380. PMID:11237598.
  4. Du X et al. (2001), Biochemistry, 40, 14166-14172. Crystal Structure and Mechanism of Catalysis of a Pyrazinamidase fromPyrococcus horikoshii†. DOI:10.1021/bi0115479. PMID:11714269.
  5. Zajc A et al. (1996), J Mol Biol, 263, 269-283. Crystallographic and Fluorescence Studies of Ligand Binding toN-Carbamoylsarcosine Amidohydrolase fromArthrobactersp. DOI:10.1006/jmbi.1996.0574. PMID:8913306.
  6. Romão MJ et al. (1992), J Mol Biol, 226, 1111-1130. Crystal structure analysis, refinement and enzymatic reaction mechanism of N-carbamoylsarcosine amidohydrolase from Arthrobacter sp. at 2·0Åresolution. DOI:10.1016/0022-2836(92)91056-u. PMID:1381445.

Step 1. Asp51 deprotonates Cys177, which initiates a nucleophilic attack on the amide carbon of N-carbamoylsarcosine in an addition reaction forming an oxyanion.

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Catalytic Residues Roles

Residue Roles
Cys177A hydrogen bond donor
Asp51A hydrogen bond acceptor
Lys144A hydrogen bond donor, electrostatic stabiliser, activator
Thr173A hydrogen bond donor, electrostatic stabiliser
Ala172A (main-C) hydrogen bond acceptor, electrostatic stabiliser
Cys177A nucleophile
Asp51A proton acceptor
Cys177A proton donor

Chemical Components

ingold: bimolecular nucleophilic addition, overall reactant used, enzyme-substrate complex formation, intermediate formation, proton transfer

Step 2. The oxyanion collapses, eliminating ammonia, which deprotonates the Asp51.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys177A covalently attached
Asp51A hydrogen bond donor, hydrogen bond acceptor
Lys144A electrostatic stabiliser, hydrogen bond donor
Thr173A hydrogen bond donor, electrostatic stabiliser
Ala172A (main-C) hydrogen bond acceptor, electrostatic stabiliser
Asp51A proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, overall product formed, deamination, enzyme-substrate complex cleavage, intermediate collapse, intermediate formation, proton transfer

Step 3. Asp51 deprotonates water, which initiates a nucleophilic attack on the carbonyl carbon of the covalently attached intermediate in an addition reaction, forming an oxyanion.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys177A covalently attached
Asp51A hydrogen bond acceptor
Lys144A hydrogen bond donor
Thr173A hydrogen bond donor
Asp51A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, overall reactant used, enzyme-substrate complex formation, intermediate formation, proton transfer

Step 4. The oxyanion collapses, eliminating Cys177, which deprotonates the Asp51.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys177A hydrogen bond acceptor
Asp51A hydrogen bond donor
Lys144A hydrogen bond donor, electrostatic stabiliser
Thr173A hydrogen bond donor, electrostatic stabiliser
Cys177A proton acceptor
Asp51A proton donor
Cys177A nucleofuge

Chemical Components

ingold: unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, intermediate collapse, intermediate terminated, overall product formed, native state of enzyme regenerated, proton transfer

Step 5. The product spontaneously decarboxylates.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, proton transfer
Download: Image, Marvin File

Step 1. Asp51 deprotonates Cys177, which initiates a nucleophilic attack on the amide carbon of N-carbamoylsarcosine in an addition reaction forming an oxyanion.

Step 2. The oxyanion collapses, eliminating ammonia, which deprotonates the Asp51.

Step 3. Asp51 deprotonates water, which initiates a nucleophilic attack on the carbonyl carbon of the covalently attached intermediate in an addition reaction, forming an oxyanion.

Step 4. The oxyanion collapses, eliminating Cys177, which deprotonates the Asp51.

Step 5. The product spontaneously decarboxylates.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Sophie T. Williams, Alex Gutteridge, Craig Porter, Katherine Ferris