4-chlorobenzoyl coenzyme A dehalogenase - Pseudomonas sp. (strain CBS-3)

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A5JTM5 (CBADH_PSEUC) Reviewed, UniProtKB/Swiss-Prot

Last modified April 5, 2011. Version 20. History...

Clusters with 100%, 90%, 50% identity |

Documents (3) |

Third-party data

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Names·Attributes·General annotation·Ontologies·Sequence annotation·Sequences·References·Cross-refs·Entry info·Documents Customize order

Names and origin

Protein names	Recommended name: 4-chlorobenzoyl coenzyme A dehalogenase Short name=4-CBA-CoA dehalogenase Short name=4-CBCoA dehalogenase Short name=4-chlorobenzoyl-CoA dehalogenase EC=3.8.1.7
Organism	Pseudomonas sp. (strain CBS-3)
Taxonomic identifier	72586 [NCBI]
Taxonomic lineage	Bacteria › Proteobacteria

Protein attributes

Sequence length	269 AA.
Sequence status	Complete.
Protein existence	Evidence at protein level

General annotation (Comments)

Function	Dehalogenates 4-chlorobenzoyl-CoA, 4-iodobenzoyl-CoA and 4-bromobenzoyl-CoA, but not 4-fluorobenzoyl-CoA. Inactive towards crotonyl-CoA, alpha-methylcrotonyl-CoA and beta-methylcrotonyl-CoA. Ref.3 Ref.4 Ref.5
Catalytic activity	4-chlorobenzoyl-CoA + H₂O = 4-hydroxybenzoyl CoA + chloride. Ref.3 Ref.4 Ref.5
Enzyme regulation	Inactivated by 1 mM Ag⁺ and by 5 mM Cu²⁺. Partially inhibited by 5 mM Zn²⁺, Mn²⁺, Co²⁺, Fe²⁺ and Ni²⁺. Unaffected by 10 mM Na⁺, K⁺ and Li⁺ and by 0.5 mM Mg²⁺, Mn²⁺, Fe²⁺, Ca²⁺, Co²⁺ and Zn²⁺. Inhibited by the sulfhydryl blocking agent 5,5'-dithio-bis-(2-nitrobenzoate), SDS and N-bromosuccinimide. Unaffected by sodium azide and EDTA. Inactivated following treatment with diethyl pyrocarbonate; this inactivation is reversible by treatment with hydroxylamine. Ref.3 Ref.4 Ref.5 Ref.6
Pathway	Xenobiotic degradation; 4-chlorobenzoate degradation; 4-hydroxybenzoate from 4-chlorobenzoate: step 2/3. Ref.4
Subunit structure	Homotetramer (Ref.4). Homotetramer, homooctamer and larger multimers (Ref.3). Homotrimer (Ref.11). Ref.3 Ref.4 Ref.5 Ref.9 Ref.11
Induction	By 4-chlorobenzoyl-CoA, 4-iodobenzoyl-CoA or 4-bromobenzoyl-CoA. Ref.3 Ref.4 Ref.5 Ref.6
Sequence similarities	Belongs to the enoyl-CoA hydratase/isomerase family.
Biophysicochemical properties	Kinetic parameters: K_M=3.7 µM for 4-chlorobenzoyl-CoA Ref.3 Ref.4 Ref.5 Ref.7 Ref.8 Ref.9 Ref.10 Ref.12 K_M=4.2 µM for 4-bromobenzoyl-CoA Ref.5 K_M=6.5 µM for 4-iodobenzoyl-CoA Ref.5 K_M=10.4 µM for 2,4-dichlorobenzoyl-CoA Ref.5 K_M=42 µM for 3,4-dichlorobenzoyl-CoA Ref.5 K_M=30 µM for 4-chloro-2-nitrobenzoyl-CoA Ref.5 K_M=5.5 µM for 4-chloro-3-nitrobenzoyl-CoA Ref.5 V_max=2.2 µmol/min/mg enzyme with 4-chlorobenzoyl-CoA as substrate Ref.5 pH dependence: Optimum pH is 10.0. Half maximum activity remains at pH 9.0 and 11.5. Stable from pH 6.5 to 11.0, activity is lost following 10 minutes incubation at pH 4.5 or 12.0. Ref.5 Temperature dependence: Optimum temperature is 60 degrees Celsius. Retains 60% of maximum activity at 30 degrees Celsius and 65 degrees Celsius. After 15 minutes preincubation at 60 degrees Celsius 70% of the initial activity remains, 15 minutes preincubation at 65 degrees Celsius results in a total loss of activity. Ref.5

Ontologies

Keywords
Molecular function	Hydrolase
Technical term	3D-structure Direct protein sequencing
Gene Ontology (GO)
Biological process	coenzyme A metabolic process Inferred from direct assay Ref.3. Source: UniProtKB
Molecular function	4-chlorobenzoyl-CoA dehalogenase activity Inferred from direct assay Ref.3. Source: UniProtKB
Complete GO annotation...

Sequence annotation (Features)

Feature key

Position(s)

Length

Description

Graphical view

Feature identifier

Molecule processing

Chain

1 – 269

269

4-chlorobenzoyl coenzyme A dehalogenase

PRO_0000401147

Regions

Region

62 – 67

Substrate binding

Sites

Active site

Proton acceptor Ref.6 Ref.9 Ref.11

Active site

145

Nucleophile Ref.6 Ref.9 Ref.11

Binding site

Substrate

Binding site

114

Substrate; via amide nitrogen

Binding site

257

Substrate; shared with oligomeric partner

Experimental info

Mutagenesis

R → K or L: Does not strongly affect catalytic activity, but reduces substrate CoA binding. Ref.8 Ref.12

Mutagenesis

E → T: Forms inclusion bodies. Ref.6

Mutagenesis

E → A: No effect on catalytic activity. Ref.6

Mutagenesis

D → A: No effect on catalytic activity. Ref.6

Mutagenesis

D → A: No effect on catalytic activity. Ref.6

Mutagenesis

G → A, I or P: Yields insoluble protein. Ref.9

Mutagenesis

F → A: 30-fold reduction in catalytic activity, substrate benzoyl group binding is unaffected. Ref.7 Ref.8 Ref.9 Ref.12

Mutagenesis

F → L: Retains catalytic activity, but substrate benzoyl group binding is decreased. Ref.7 Ref.8 Ref.9 Ref.10

Mutagenesis

F → P: Severely reduces catalytic activity. Arylated intermediate does not accumulate. Ref.7 Ref.8 Ref.9 Ref.12

Mutagenesis

Y → D: Catalytic activity is almost as efficient as wild type. Ref.9

Mutagenesis

R → K: Reduces substrate CoA binding. Ref.8 Ref.12

Mutagenesis

R → L: Forms inclusion bodies. Ref.8 Ref.12

Mutagenesis

E → T: No effect on catalytic activity. Ref.6

Mutagenesis

H → Q: Loss of catalytic activity, substrate benzoyl group binding is not affected. Ref.6 Ref.7 Ref.10

Mutagenesis

F → L: Retains catalytic activity, but substrate benzoyl group binding is decreased. Ref.7 Ref.10

Mutagenesis

W → F: Retains catalytic activity, but substrate benzoyl group binding is decreased. Ref.7 Ref.10

Mutagenesis

W → Y: Reduced activity and substrate benzoyl group binding. Ref.7 Ref.10

Mutagenesis

H → Q: Complete loss of catalytic activity (PubMed:8718880 and PubMed:9063883). Significantly reduced activity (PubMed:11695894). Substrate binding is not significantly affected. Reduced arylated intermediate formation. Ref.6 Ref.7 Ref.10 Ref.12

Mutagenesis

H → Q: No effect on catalytic activity. Ref.6

Mutagenesis

112

A → G: Yields insoluble protein. Ref.9

Mutagenesis

112

A → S: Protein precipitates upon purification. Ref.9

Mutagenesis

112

A → V: Catalytic activity is almost as efficient as wild type. Ref.9

Mutagenesis

113

G → A: Strongly reduced catalytic activity and substrate benzoyl group binding. Arylated intermediate does not accumulate. Ref.8 Ref.9 Ref.12

Mutagenesis

113

G → N or S: Strongly reduced catalytic activity. Arylated intermediate does not accumulate. Ref.8 Ref.9

Mutagenesis

113

G → V: Protein precipitates upon purification. Ref.8 Ref.9

Mutagenesis

114

G → A: Strongly reduced catalytic activity and substrate benzoyl group binding. Ref.7 Ref.8 Ref.10 Ref.12

Mutagenesis

114

G → P: Unstable. Ref.7 Ref.8 Ref.10

Mutagenesis

115

G → L, N, S or V: Yields insoluble protein. Ref.9

Mutagenesis

123

D → T: No effect on catalytic activity. Ref.6

Mutagenesis

129

D → T: No effect on catalytic activity. Ref.6

Mutagenesis

137

W → F: Low catalytic activity, but KM unaffected (PubMed:8718880). Retains catalytic activity, but substrate benzoyl group binding is decreased (PubMed:9063883). Ref.6 Ref.7 Ref.10

Mutagenesis

145

D → A: Complete loss of catalytic activity, but not substrate binding. Ref.6 Ref.7 Ref.10

Mutagenesis

163

E → T: No effect on catalytic activity. Ref.6

Mutagenesis

175

E → D: No effect on catalytic activity. Ref.6

Mutagenesis

179

W → F: No effect on catalytic activity. Ref.6

Mutagenesis

208

H → Q: No effect on catalytic activity. Ref.6

Mutagenesis

216

R → E, K or L: Yields insoluble protein. Ref.10

Mutagenesis

232

E → A, N, Q or R: Yields insoluble protein. Ref.10

Mutagenesis

232

E → D: Reduced catalytic activity, increased substrate binding. Ref.10

Mutagenesis

257

R → K: Retains catalytic activity and substrate CoA binding. Ref.8 Ref.12

Mutagenesis

257

R → L: Significantly reduces catalytic activity and substrate CoA binding. Ref.8 Ref.12

Secondary structure

269

Helix Strand Turn

Details...

Sequences

Sequence

Length

Mass (Da)

Tools

A5JTM5 [UniParc].

Last modified June 26, 2007. Version 1.
Checksum: EC165E9063C70B07
Show »

FASTA

269

29,802

        10         20         30         40         50         60 
MYEAIGHRVE DGVAEITIKL PRHRNALSVK AMQEVTDALN RAEEDDSVGA VMITGAEDAF 

        70         80         90        100        110        120 
CAGFYLREIP LDKGVAGVRD HFRIGALWWH QMIHKIIRVK RPVLAAINGV AAGGGLGISL 

       130        140        150        160        170        180 
ASDMAICADS AKFVCAWHTI GIGNDTATSY SLARIVGMRR AMELMLTNRT LYPEEAKDWG 

       190        200        210        220        230        240 
LVSRVYPKDD FREVAWKVAR ELAAAPTHLQ VMAKERFHAG WMQPVEECTE FEIQNVIASV 

       250        260 
THPHFMPCLT EFLDGHRADR PQVELPAGV

« Hide

References

[1]	"Ancestry of the 4-chlorobenzoate dehalogenase: analysis of amino acid sequence identities among families of acyl:adenyl ligases, enoyl-CoA hydratases/isomerases, and acyl-CoA thioesterases." Babbitt P.C., Kenyon G.L., Martin B.M., Charest H., Slyvestre M., Scholten J.D., Chang K.H., Liang P.H., Dunaway-Mariano D. Biochemistry 31:5594-5604(1992) [PubMed: 1351742] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA].
[2]	"The nucleotide sequence upstream and downstream of 4-CBA-CoA ORFs in 9.5 kb Pseudomonas sp. strain CBS 3 chromosomal DNA." Zhang W., Dunaway-Mariano D. Submitted (APR-2007) to the EMBL/GenBank/DDBJ databases Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA]. Strain: CBS-3.
[3]	"Dehalogenation of 4-chlorobenzoate. Characterisation of 4-chlorobenzoyl-coenzyme A dehalogenase from Pseudomonas sp. CBS3." Loffler F., Lingens F., Muller R. Biodegradation 6:203-212(1995) [PubMed: 7579994] [Abstract] Cited for: PROTEIN SEQUENCE OF 1-40, FUNCTION, CATALYTIC ACTIVITY, ENZYME REGULATION, BIOPHYSICOCHEMICAL PROPERTIES, SUBUNIT, INDUCTION. Strain: CBS-3.
[4]	"Isolation and characterization of the three polypeptide components of 4-chlorobenzoate dehalogenase from Pseudomonas sp. strain CBS-3." Chang K.H., Liang P.H., Beck W., Scholten J.D., Dunaway-Mariano D. Biochemistry 31:5605-5610(1992) [PubMed: 1610806] [Abstract] Cited for: FUNCTION, CATALYTIC ACTIVITY, ENZYME REGULATION, BIOPHYSICOCHEMICAL PROPERTIES, PATHWAY, SUBUNIT. Strain: CBS-3.
[5]	"Specificity of 4-chlorobenzoyl coenzyme A dehalogenase catalyzed dehalogenation of halogenated aromatics." Liang P.H., Yang G., Dunaway-Mariano D. Biochemistry 32:12245-12250(1993) [PubMed: 8218302] [Abstract] Cited for: BIOPHYSICOCHEMICAL PROPERTIES.
[6]	"Identification of active site residues essential to 4-chlorobenzoyl-coenzyme A dehalogenase catalysis by chemical modification and site directed mutagenesis." Yang G., Liu R.Q., Taylor K.L., Xiang H., Price J., Dunaway-Mariano D. Biochemistry 35:10879-10885(1996) [PubMed: 8718880] [Abstract] Cited for: ENZYME REGULATION, ACTIVE SITES, MUTAGENESIS OF GLU-34; GLU-43; ASP-45; ASP-46; GLU-68; HIS-90; HIS-94; ASP-123; ASP-129; TRP-137; GLU-163; GLU-175; TRP-179 AND HIS-208.
[7]	"Investigation of substrate activation by 4-chlorobenzoyl-coenzyme A dehalogenase." Taylor K.L., Xiang H., Liu R.Q., Yang G., Dunaway-Mariano D. Biochemistry 36:1349-1361(1997) [PubMed: 9063883] [Abstract] Cited for: BIOPHYSICOCHEMICAL PROPERTIES, MUTAGENESIS OF PHE-64; HIS-81; PHE-82; TRP-89; HIS-90; GLY-114; TRP-137 AND ASP-145.
[8]	"Role of active site binding interactions in 4-chlorobenzoyl-coenzyme A dehalogenase catalysis." Luo L., Taylor K.L., Xiang H., Wei Y., Zhang W., Dunaway-Mariano D. Biochemistry 40:15684-15692(2001) [PubMed: 11747444] [Abstract] Cited for: BIOPHYSICOCHEMICAL PROPERTIES, MUTAGENESIS OF ARG-24; PHE-64; ARG-67; GLY-113; GLY-114 AND ARG-257.
[9]	"The strength of dehalogenase-substrate hydrogen bonding correlates with the rate of Meisenheimer intermediate formation." Dong J., Lu X., Wei Y., Luo L., Dunaway-Mariano D., Carey P.R. Biochemistry 42:9482-9490(2003) [PubMed: 12899635] [Abstract] Cited for: BIOPHYSICOCHEMICAL PROPERTIES, MUTAGENESIS OF GLY-63; PHE-64; TYR-65; ALA-112; GLY-113 AND GLY-115.
[10]	"Contributions of long-range electrostatic interactions to 4-chlorobenzoyl-CoA dehalogenase catalysis: a combined theoretical and experimental study." Wu J., Xu D., Lu X., Wang C., Guo H., Dunaway-Mariano D. Biochemistry 45:102-112(2006) [PubMed: 16388585] [Abstract] Cited for: BIOPHYSICOCHEMICAL PROPERTIES, MUTAGENESIS OF ARG-216 AND GLU-232.
[11]	"Structure of 4-chlorobenzoyl coenzyme A dehalogenase determined to 1.8 A resolution: an enzyme catalyst generated via adaptive mutation." Benning M.M., Taylor K.L., Liu R.-Q., Yang G., Xiang H., Wesenberg G., Dunaway-Mariano D., Holden H.M. Biochemistry 35:8103-8109(1996) [PubMed: 8679561] [Abstract] Cited for: X-RAY CRYSTALLOGRAPHY (1.80 ANGSTROMS) OF MUTANT GLN-90 IN COMPLEX WITH 4-HYDROXYBENZOYL COENZYME A, SUBUNIT, ACTIVE SITE, SUBSTRATE-BINDING SITES. Strain: CBS-3.
[12]	"Histidine 90 function in 4-chlorobenzoyl-coenzyme a dehalogenase catalysis." Zhang W., Wei Y., Luo L., Taylor K.L., Yang G., Dunaway-Mariano D., Benning M.M., Holden H.M. Biochemistry 40:13474-13482(2001) [PubMed: 11695894] [Abstract] Cited for: X-RAY CRYSTALLOGRAPHY (1.90 ANGSTROMS) OF MUTANT GLN-90 IN COMPLEX WITH 4-HYDROXYBENZOYL COENZYME A, BIOPHYSICOCHEMICAL PROPERTIES, MUTAGENESIS OF HIS-90.

Cross-references

Sequence databases

EMBL
GenBank
DDBJ

EF569604 Genomic DNA. Translation: ABQ44578.1.

3D structure databases

PDBe
RCSB PDB
PDBj

Entry	Method	Resolution (Å)	Chain	Positions	PDBsum
1JXZ	X-ray	1.90	A/B/C	1-269	[»]
1NZY	X-ray	1.80	A/B/C	1-269	[»]

ProteinModelPortal

A5JTM5.

SMR

A5JTM5. Positions 1-269.

ModBase

Search...

Protocols and materials databases

StructuralBiologyKnowledgebase

Search...

Enzyme and pathway databases

BioCyc

MetaCyc:MONOMER-14753.

Family and domain databases

InterPro

IPR014748. Crontonase_C.
IPR001753. Crotonase_core.
IPR018376. Enoyl-CoA_hyd/isom_CS.
[Graphical view]

Gene3D

G3DSA:1.10.12.10. Crontonase_C. 1 hit.

Pfam

PF00378. ECH. 1 hit.
[Graphical view]

PROSITE

PS00166. ENOYL_COA_HYDRATASE. 1 hit.
[Graphical view]

ProtoNet

Search...

Entry information

Entry name

CBADH_PSEUC

Accession

Primary (citable) accession number: A5JTM5

Entry history

Integrated into UniProtKB/Swiss-Prot:	November 30, 2010
Last sequence update:	June 26, 2007
Last modified:	April 5, 2011
This is version 20 of the entry and version 1 of the sequence. [Complete history]

Entry status

Reviewed (UniProtKB/Swiss-Prot)

Annotation program

Prokaryotic Protein Annotation Program

Relevant documents

PATHWAY comments

Index of metabolic and biosynthesis pathways

PDB cross-references

Index of Protein Data Bank (PDB) cross-references

SIMILARITY comments

Index of protein domains and families

Names·Attributes·General annotation·Ontologies·Sequence annotation·Sequences·References·Cross-refs·Entry info·Documents

Preferred order of sections

Names and origin

Protein attributes

General annotation (Comments)

Ontologies

Sequence annotation (Features)

Molecule processing

Regions

Sites

Experimental info

Secondary structure

Sequences

References

Cross-references

Sequence databases

3D structure databases

Protocols and materials databases

Enzyme and pathway databases

Family and domain databases

Entry information

Relevant documents