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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

Regulation summary diagram for mhpC

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 [Burlingame83a].

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, Henderson97a, Fleming00, Li06b]. Analysis of site-directed mutant enzymes suggests that Ser110 activates the nucleophilic water molecule [Li05a]. 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 [Li06c]. Esterase, thioesterase, and hydroxamate formation activities of the wild type and various mutant enzymes has been measured [Li08a].

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: [Ferrandez97a]

Locations: cytosol

Map Position: [370,463 -> 371,329] (7.98 centisomes, 29°)
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, InterPro:IN-FAMILY:IPR029058, PDB:Structure:1U2E, Pfam:IN-FAMILY:PF12697, Prints:IN-FAMILY:PR00111, Prints:IN-FAMILY:PR00412

Gene-Reaction Schematic

Gene-Reaction Schematic

Genetic Regulation Schematic

Genetic regulation schematic for mhpC


GO Terms:
Biological Process:
Inferred from experimentInferred by computational analysisGO:0019439 - aromatic compound catabolic process [UniProtGOA11a, GOA06, GOA01a, Burlingame86]
Inferred from experimentGO:0019622 - 3-(3-hydroxy)phenylpropionate catabolic process [Burlingame86]
Inferred by computational analysisGO:0019380 - 3-phenylpropionate catabolic process [UniProtGOA12]
Molecular Function:
Inferred from experimentInferred by computational analysisGO:0016787 - hydrolase activity [UniProtGOA11a, Lam97, Li05a]
Inferred from experimentInferred by computational analysisGO:0018771 - 2-hydroxy-6-oxonona-2,4-dienedioate hydrolase activity [GOA06, GOA01, GOA01a, Lam97]
Inferred from experimentInferred by computational analysisGO:0052823 - 2-hydroxy-6-oxonona-2,4,7-trienedioate hydrolase activity [GOA01, Lam97]
Inferred by computational analysisGO:0003824 - catalytic activity [GOA01a]
Inferred by computational analysisGO:0042803 - protein homodimerization activity [GOA01a]
Cellular Component:
Inferred from experimentInferred by computational analysisGO:0005737 - cytoplasm [GOA01a, Lam97]
Inferred by computational analysisGO:0005829 - cytosol [DiazMejia09]

MultiFun Terms: metabolismcarbon utilizationcarbon compounds

Essentiality data for mhpC knockouts:

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enrichedYes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB LennoxYes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerolYes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucoseYes 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)

Inferred from experiment

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 direction shown, i.e. which substrates are on the left and right sides, 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-phenylpropanoate and 3-(3-hydroxyphenyl)propanoate 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 17.14 [Lam97]

pH(opt): 6.5-9.5 [Lam97]


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

Inferred from experiment

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 direction shown, i.e. which substrates are on the left and right sides, 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 0.38 [Lam97]

Sequence Features

Protein sequence of 2-hydroxy-6-ketonona-2,4-dienedioate hydrolase with features indicated

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
Inferred from experiment[Lam97, Lam97, Lam97]
 
Chain 2 -> 288
Author statement[UniProt15]
UniProt: 2-hydroxy-6-oxononadienedioate/2-hydroxy-6-oxononatrienedioate hydrolase.
Mutagenesis-Variant 44
Inferred from experiment[Li05a]
UniProt: 2-fold decrease in catalytic efficiency and more than 5-fold increase in affinity for the natural substrate.
Mutagenesis-Variant 113
Inferred from experiment[Li06c]
Inferred from experiment[Li06c]
N → A: 200-fold decrease in catalytic activity and almost 2-fold increase in affinity.
N → H: 350-fold decrease in catalytic activity and almost 2-fold increase in affinity.
Mutagenesis-Variant 114
Inferred from experiment[Li05a]
S → A or G: Weakly active. 3-fold decrease in affinity. Fast ketonisation and slow C-C cleavage.
Amino-Acid-Site 114
Author statement[UniProt15]
UniProt: Transition state stabilizer; Sequence Annotation Type: site.
Mutagenesis-Variant 118
Inferred from experiment[Li05a]
UniProt: More than 2-fold decrease in catalytic efficiency and 3-fold increase affinity.
Sequence-Conflict 153
Inferred by curator[UniProt15]
UniProt: (in Ref. 1; BAA13054).
Mutagenesis-Variant 177
Inferred from experiment[Li06c]
Inferred from experiment[Li06c]
F → D: 100-fold decrease in catalytic activity.
F → G: 4-fold and 8-fold decrease in catalytic activity and affinity, respectively.
Mutagenesis-Variant 192
Inferred from experiment[Li06c]
Inferred from experiment[Li06c]
R → Q: 280-fold and 10-fold decrease in catalytic activity and affinity, respectively.
R → K: 40-fold and 5-fold decrease in catalytic activity and affinity, respectively.
Amino-Acid-Site 192
Author statement[UniProt15]
UniProt: Catalytic role in ketonization of the dienol substrate (substrate destabilization); Sequence Annotation Type: site.
Mutagenesis-Variant 265
Inferred from experiment[Li06c]
UniProt: 2-fold decrease in catalytic activity and almost 2-fold increase in affinity.
Mutagenesis-Variant 267
Inferred from experiment[Li05a]
UniProt: Weakly active, 1000-fold decrease in catalytic efficiency. Very slow ketonisation and C-C cleavage.
Active-Site 267
Author statement[UniProt15]
UniProt: Proton acceptor.
Mutagenesis-Variant 268
Inferred from experiment[Li06c]
UniProt: 10-fold and 20-fold decrease in catalytic activity and affinity, respectively.


Sequence Pfam Features

Protein sequence of 2-hydroxy-6-ketonona-2,4-dienedioate hydrolase with features indicated

Feature Class Location Citations Comment
Pfam PF12697 38 -> 279
Inferred by computational analysis[Finn14]
Abhydrolase_6 : Alpha/beta hydrolase family


Gene Local Context (not to scale -- see Genome Browser for correct scale)

Gene local context diagram

Transcription Unit

Transcription-unit diagram

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

Burlingame83a: 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

Ferrandez97a: 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

Finn14: Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, Heger A, Hetherington K, Holm L, Mistry J, Sonnhammer EL, Tate J, Punta M (2014). "Pfam: the protein families database." Nucleic Acids Res 42(Database issue);D222-30. PMID: 24288371

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

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

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

Henderson97a: 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

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

Li05a: 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

Li06b: 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

Li06c: 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

Li08a: 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

UniProt15: UniProt Consortium (2015). "UniProt version 2015-08 released on 2015-07-22." Database.

UniProtGOA11a: 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
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