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CSA entry for 1uk7
Original Entry
Title:
Hydrolase
Compound:
Crystal structure of a meta-cleavage product hydrolase
(cumd) complexed with n-butyrate
Mutant:
Yes
UniProt/Swiss-Prot:
P96965-P96965
EC Class:
3.7.1.9
Other CSA Entries:
Overview of all sites for 1uk7
Homologues of 1uk7
Entries for UniProt/Swiss-Prot: P96965
Entries for EC: 3.7.1.9
Other Databases:
PDB entry: 1uk7
PDBsum entry: 1uk7
UniProt/Swiss-Prot: P96965
IntEnz entry: 3.7.1.9
Literature Report:
Introduction:
Meta-cleavage product hydrolases (MCP-hydrolase) are key enzymes involved in the microbial degradation of aromatic compounds. MCP-hydrolase CumD is a dimeric enzyme produced by
Pseudomonas fluorescens IP01 and is involved in the pathway for the degradation of cumene. MCP-hydrolase enzymes produce 2-hydroxypenta-2,4-dienoate and various organic acids,
depending upon the C6 substituent of the substrate. CumD prefers larger C6 substituents compared to other monoalkylbenzene hydrolases, such as TodF. Hence, when CumD acts on the meta-cleavage product of cumene, 2-hydroxypenta-2,4-dienoate and isobutyric acid are the products.

The understanding of the catalytic mechanism of MCP-hydrolases is neccessary to improve biological degradation of environmental pollutant aromatic compounds, which may be carcinogenic, toxic and mutagenic.

Mechanism:
The active site of CumD contains the Ser103, Asp 224, His 252 catalytic triad. Studies have shown that reaction proceeds not through initial nucleophilic attack, but via base catalysed attack on water by Ser 103.

1. The His 252 deprotonates Ser 103, activating it to basic attack of a water molecule.
2. The Ser 103 deprotonates the water, which then attacks the carbonyl carbon of the substrate.
3. The His and Ser residues form an active site ion pair, which is stabilised by the Asp 224 residue.
4. Hydrolysis of the substrate occurs, cleaving the C-C bond

Sites:

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Found by:
Literature reference 

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ALAA 103 103Sidechain
Acid/baseResidue
Acid/baseWater
ElectrostaticResidue
Ser 103 is deprotonated by his 252, and so can then act as a general base on a water molecule, activating it towards nucleophilic attack of the substrate carbonyl carbon. The negatively charged side chain Ser oxygen and the His imidazole ring form an active site ion pair.
Evidence from paper Evidence concerns Evidence type
PubMed ID 15784976 Current protein Mutagenesis of residue
PubMed ID 16233251 Current protein Mutagenesis of residue
PubMed ID 16233251 Current protein Structural similarity to homologue of known mechanism
PubMed ID 16233251 Current protein Conservation of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ASPA 224 224Sidechain
ElectrostaticResidue
The Asp 224 forms a hydrogen bond to the His 252 to stabilise the ion pair.
Evidence from paper Evidence concerns Evidence type
PubMed ID 16233251 Current protein Mutagenesis of residue
PubMed ID 16233251 Current protein Structural similarity to homologue of known mechanism
PubMed ID 16233251 Current protein Conservation of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
HISA 252 252Sidechain
ElectrostaticResidue
Acid/baseResidue
The his 252 deprotonates an adjacent serine residue and also forms an active site ion pair with the charged serine to stabilise the transition state.
Evidence from paper Evidence concerns Evidence type
PubMed ID 16233251 Current protein Mutagenesis of residue
PubMed ID 16233251 Current protein Conservation of residue
Notes:
The Pseudomonas fluorescens species is able to grow on cumene and toluene as the sole source of carbon, but it cannot grow on biphenyl because the meta-cleavage pathway is blocked at the CumD step.

Ile and Trp 143 on the helix alpha-4 appear to cause steric hinderance with the aromatic ring of the substrate, hampering base-catalysed attack by water.

References:
1
A series of crystal structures of a meta-cleavage product hydrolase from Pseudomonas fluorescens IP01 (CumD) complexed with various cleavage products.
S. Fushinobu and S. Y. Jun and M. Hidaka and H. Nojiri and H. Yamane and H. Shoun and T. Omori and T. Wakagi
Biosci Biotechnol Biochem 69, (3) 491-8, (2005).
15784976
2
Purification, characterization, and steady-state kinetics of a meta-cleavage compound hydrolase from Pseudomonas fluorescens IPO1.
T. Saku and S. Fushinobu and S. Y. Jun and N. Ikeda and H. Nojiri and H. Yamane and T. Omori and T. Wakagi
J Biosci Bioeng 93, (6) 568-74, (2002).
16233251
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