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Escherichia coli K-12 substr. MG1655 Enzyme: 2-hydroxy-6-oxononatrienedioate hydrolase



Gene: mhpC Accession Numbers: M012 (EcoCyc), b0349, ECK0346

Regulation Summary Diagram: ?

Subunit composition of 2-hydroxy-6-oxononatrienedioate hydrolase = [MhpC]2
         2-hydroxy-6-ketonona-2,4-dienedioate hydrolase = MhpC

Summary:
2-hydroxy-6-oxononatrienedioate hydrolase (MhpC) catalyzes the hydrolysis of the ring fission product in the meta-cleavage pathway for the catabolism of 3-(3-hydroxyphenyl)propionate [Burlingame83].

The catalytic mechanism has been investigated. The first catalytic step appears to be a reversible keto-enol tautomerization, followed by a base-catalyzed attack of water forming a gem-diol intermediate [Lam97, Henderson97c, Fleming00, Li06i]. Analysis of site-directed mutant enzymes suggests that Ser110 activates the nucleophilic water molecule [Li05e]. Arg188 has a catalytic role in ketonization of the dienol substrate; Phe173 and Trp264 appear to be involved in substrate binding, while Asn109 is positioning the active site loop containing Ser110 [Li06j]. Esterase, thioesterase, and hydroxamate formation activities of the wild type and various mutant enzymes has been measured [Li08h].

A crystal structure of MhpC has been solved at 2.1 Å resolution [Dunn05].

An mhpC mutant does not grow with m-hydroxyphenylpropionate (MHP) or 3-phenylpropionate as the sole source of carbon [Burlingame86].

Gene Citations: [Ferrandez97]

Locations: cytosol

Map Position: [370,463 -> 371,329] (7.98 centisomes)
Length: 867 bp / 288 aa

Molecular Weight of Polypeptide: 31.937 kD (from nucleotide sequence), 29.0 kD (experimental) [Lam97 ]

Molecular Weight of Multimer: 62.0 kD (experimental) [Lam97]

Unification Links: ASAP:ABE-0001201 , DIP:DIP-10207N , EchoBASE:EB4168 , EcoGene:EG20275 , EcoliWiki:b0349 , ModBase:P77044 , OU-Microarray:b0349 , PortEco:mhpC , PR:PRO_000023230 , Pride:P77044 , Protein Model Portal:P77044 , RefSeq:NP_414883 , RegulonDB:M012 , SMR:P77044 , String:511145.b0349 , UniProt:P77044

Relationship Links: InterPro:IN-FAMILY:IPR000073 , InterPro:IN-FAMILY:IPR000639 , InterPro:IN-FAMILY:IPR023791 , PDB:Structure:1U2E , Pfam:IN-FAMILY:PF00561 , Prints:IN-FAMILY:PR00111 , Prints:IN-FAMILY:PR00412

Gene-Reaction Schematic: ?

Genetic Regulation Schematic: ?

GO Terms:

Biological Process: GO:0019439 - aromatic compound catabolic process Inferred from experiment Inferred by computational analysis [UniProtGOA11, GOA06, GOA01, Burlingame86]
GO:0019622 - 3-(3-hydroxy)phenylpropionate catabolic process Inferred from experiment [Burlingame86]
GO:0019380 - 3-phenylpropionate catabolic process Inferred by computational analysis [UniProtGOA12]
Molecular Function: GO:0016787 - hydrolase activity Inferred from experiment Inferred by computational analysis [UniProtGOA11, Lam97, Li05e]
GO:0018771 - 2-hydroxy-6-oxonona-2,4-dienedioate hydrolase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, GOA01, Lam97]
GO:0052823 - 2-hydroxy-6-oxonona-2,4,7-trienedioate hydrolase activity Inferred from experiment Inferred by computational analysis [GOA01a, Lam97]
GO:0003824 - catalytic activity Inferred by computational analysis [GOA01]
GO:0042803 - protein homodimerization activity Inferred by computational analysis [GOA01]
Cellular Component: GO:0005737 - cytoplasm Inferred from experiment Inferred by computational analysis [GOA01, Lam97]
GO:0005829 - cytosol Inferred by computational analysis [DiazMejia09]

MultiFun Terms: metabolism carbon utilization carbon compounds

Essentiality data for mhpC knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
Yes [Feist07, Comment 4]

Credits:
Created 27-Oct-2011 by Keseler I , SRI International
Last-Curated ? 27-Oct-2011 by Keseler I , SRI International


Enzymatic reaction of: 2-hydroxy-6-ketonona-2,4-dienedioate hydrolase (2-hydroxy-6-oxononatrienedioate hydrolase)

EC Number: 3.7.1.14

(2Z,4E)-2-hydroxy-6-oxonona-2,4-diene-1,9-dioate + H2O <=> (2Z)-2-hydroxypenta-2,4-dienoate + succinate + H+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is irreversible in the direction shown. [Lam97]

Alternative Substrates [Comment 5]:

In Pathways: 3-phenylpropionate and 3-(3-hydroxyphenyl)propionate degradation , 3-phenylpropanoate and 3-(3-hydroxyphenyl)propanoate degradation to 2-oxopent-4-enoate

Inhibitors (Unknown Mechanism): p-hydroxymercuribenzoate [Lam97]

Kinetic Parameters:

Substrate
Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Citations
(2Z,4E)-2-hydroxy-6-oxonona-2,4-diene-1,9-dioate
2.1
36.0
[Lam97]

pH(opt): 6.5-9.5 [Lam97]


Enzymatic reaction of: 2-hydroxy-6-oxononatrienedioate hydrolase

EC Number: 3.7.1.14

(2Z,4E,7E)-2-hydroxy-6-oxonona-2,4,7-triene-1,9-dioate + H2O <=> (2Z)-2-hydroxypenta-2,4-dienoate + fumarate + H+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

The reaction is favored in the direction shown.

In Pathways: cinnamate and 3-hydroxycinnamate degradation to 2-oxopent-4-enoate

Kinetic Parameters:

Substrate
Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Citations
(2Z,4E,7E)-2-hydroxy-6-oxonona-2,4,7-triene-1,9-dioate
2.9
1.1
[Lam97]


Sequence Features

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
[Lam97, Lam97]
 
Chain 2 -> 288
[UniProt12]
UniProt: 2-hydroxy-6-oxononadienedioate/2-hydroxy- 6-oxononatrienedioate hydrolase.
Mutagenesis-Variant 44
[Li05e, UniProt12]
Alternate sequence: S → A; UniProt: 2-fold decrease in catalytic efficiency and more than 5-fold increase in affinity for the natural substrate.
Mutagenesis-Variant 113
[Li06j, UniProt12]
Alternate sequence: N → H; UniProt: 350-fold decrease in catalytic activity and almost 2-fold increase in affinity.
Alternate sequence: N → A; UniProt: 200-fold decrease in catalytic activity and almost 2-fold increase in affinity.
Mutagenesis-Variant 114
[Li05e, UniProt12]
Alternate sequence: S → G; UniProt: Weakly active. 3-fold decrease in affinity. Fast ketonisation and slow C-C cleavage.
Alternate sequence: S → A; UniProt: Weakly active. 3-fold decrease in affinity. Fast ketonisation and slow C-C cleavage.
Amino-Acid-Site 114
[UniProt12]
UniProt: Transition state stabilizer; Sequence Annotation Type: site.
Mutagenesis-Variant 118
[Li05e, UniProt12]
Alternate sequence: H → A; UniProt: More than 2-fold decrease in catalytic efficiency and 3-fold increase affinity.
Sequence-Conflict 153
[Kawamukai96, UniProt12]
Alternate sequence: E → G; UniProt: (in Ref. 1; BAA13054).
Mutagenesis-Variant 177
[Li06j, UniProt12]
Alternate sequence: F → G; UniProt: 4-fold and 8-fold decrease in catalytic activity and affinity, respectively.
Alternate sequence: F → D; UniProt: 100-fold decrease in catalytic activity.
Mutagenesis-Variant 192
[Li06j, UniProt12]
Alternate sequence: R → Q; UniProt: 280-fold and 10-fold decrease in catalytic activity and affinity, respectively.
Alternate sequence: R → K; UniProt: 40-fold and 5-fold decrease in catalytic activity and affinity, respectively.
Amino-Acid-Site 192
[UniProt12]
UniProt: Catalytic role in ketonization of the dienol substrate (substrate destabilization); Sequence Annotation Type: site.
Mutagenesis-Variant 265
[Li06j, UniProt12]
Alternate sequence: C → A; UniProt: 2-fold decrease in catalytic activity and almost 2-fold increase in affinity.
Mutagenesis-Variant 267
[Li05e, UniProt12]
Alternate sequence: H → A; UniProt: Weakly active, 1000-fold decrease in catalytic efficiency. Very slow ketonisation and C-C cleavage.
Active-Site 267
[UniProt12]
UniProt: Proton acceptor.
Mutagenesis-Variant 268
[Li06j, UniProt12]
Alternate sequence: W → G; UniProt: 10-fold and 20-fold decrease in catalytic activity and affinity, respectively.


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
Ingrid Keseler on Mon Oct 31, 2011:
Gene start position corrected based on N-terminal sequencing results reported in [Lam97 ].
Peter D. Karp on Wed Jan 18, 2006:
Gene left-end position adjusted based on analysis performed in the 2005 E. coli annotation update [Riley06 ].
3/2/1998 (pkarp) Merged genes G6203/mhpC and M012/mhpC
Markus Krummenacker on Tue Oct 14, 1997:
Gene object created from Blattner lab Genbank (v. M52) entry.


References

Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

Burlingame83: Burlingame R, Chapman PJ (1983). "Catabolism of phenylpropionic acid and its 3-hydroxy derivative by Escherichia coli." J Bacteriol 1983;155(1);113-21. PMID: 6345502

Burlingame86: Burlingame RP, Wyman L, Chapman PJ (1986). "Isolation and characterization of Escherichia coli mutants defective for phenylpropionate degradation." J Bacteriol 1986;168(1);55-64. PMID: 3531186

Diaz98: Diaz E, Ferrandez A, Garcia JL (1998). "Characterization of the hca cluster encoding the dioxygenolytic pathway for initial catabolism of 3-phenylpropionic acid in Escherichia coli K-12." J Bacteriol 1998;180(11);2915-23. PMID: 9603882

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

Dunn05: Dunn G, Montgomery MG, Mohammed F, Coker A, Cooper JB, Robertson T, Garcia JL, Bugg TD, Wood SP (2005). "The structure of the C-C bond hydrolase MhpC provides insights into its catalytic mechanism." J Mol Biol 346(1);253-65. PMID: 15663942

Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909

Ferrandez97: Ferrandez A, Garcia JL, Diaz E (1997). "Genetic characterization and expression in heterologous hosts of the 3-(3-hydroxyphenyl)propionate catabolic pathway of Escherichia coli K-12." J Bacteriol 1997;179(8);2573-81. PMID: 9098055

Fleming00: Fleming SM, Robertson TA, Langley GJ, Bugg TD (2000). "Catalytic mechanism of a C-C hydrolase enzyme: evidence for a gem-diol intermediate, not an acyl enzyme." Biochemistry 39(6);1522-31. PMID: 10684634

Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA01a: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Henderson97c: Henderson IM, Bugg TD (1997). "Pre-steady-state kinetic analysis of 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase: kinetic evidence for enol/keto tautomerization." Biochemistry 36(40);12252-8. PMID: 9315863

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

Kawamukai96: Kawamukai M. (1996). Data submission to EMBL/GenBank/DDBJ databases on 1996-06.

Lam94: Lam WWY, Bugg TDH (1994). "Chemistry of extradiol aromatic ring cleavage: Isolation of a stable dienol ring fission intermediate and stereochemistry of its enzymatic hydrolytic cleavage." J Chem Soc, Chem Commun (1994): 1163-1164.

Lam97: Lam WW, Bugg TD (1997). "Purification, characterization, and stereochemical analysis of a C-C hydrolase: 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase." Biochemistry 36(40);12242-51. PMID: 9315862

Li05e: Li C, Montgomery MG, Mohammed F, Li JJ, Wood SP, Bugg TD (2005). "Catalytic mechanism of C-C hydrolase MhpC from Escherichia coli: kinetic analysis of His263 and Ser110 site-directed mutants." J Mol Biol 346(1);241-51. PMID: 15663941

Li06i: Li JJ, Li C, Blindauer CA, Bugg TD (2006). "Evidence for a gem-diol reaction intermediate in bacterial C-C hydrolase enzymes BphD and MhpC from 13C NMR spectroscopy." Biochemistry 45(41);12461-9. PMID: 17029401

Li06j: Li C, Li JJ, Montgomery MG, Wood SP, Bugg TD (2006). "Catalytic role for arginine 188 in the C-C hydrolase catalytic mechanism for Escherichia coli MhpC and Burkholderia xenovorans LB400 BphD." Biochemistry 45(41);12470-9. PMID: 17029402

Li08h: Li C, Hassler M, Bugg TD (2008). "Catalytic promiscuity in the alpha/beta-hydrolase superfamily: hydroxamic acid formation, C--C bond formation, ester and thioester hydrolysis in the C--C hydrolase family." Chembiochem 9(1);71-6. PMID: 18058773

Riley06: Riley M, Abe T, Arnaud MB, Berlyn MK, Blattner FR, Chaudhuri RR, Glasner JD, Horiuchi T, Keseler IM, Kosuge T, Mori H, Perna NT, Plunkett G, Rudd KE, Serres MH, Thomas GH, Thomson NR, Wishart D, Wanner BL (2006). "Escherichia coli K-12: a cooperatively developed annotation snapshot--2005." Nucleic Acids Res 34(1);1-9. PMID: 16397293

UniProt12: UniProt Consortium (2012). "UniProt version 2012-02 released on 2012-02-29 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

UniProtGOA12: UniProt-GOA (2012). "Gene Ontology annotation based on UniPathway vocabulary mapping."

Other References Related to Gene Regulation

Torres03: Torres B, Porras G, Garcia JL, Diaz E (2003). "Regulation of the mhp cluster responsible for 3-(3-hydroxyphenyl) propionic acid degradation in escherichia coli." J Biol Chem 278(30);27575-85. PMID: 12748194


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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
Page generated by SRI International Pathway Tools version 18.5 on Sun Nov 23, 2014, biocyc14.