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Escherichia coli K-12 substr. MG1655 Enzyme: NADPH-dependent curcumin/dihydrocurcumin reductase



Gene: curA Accession Numbers: G6760 (EcoCyc), b1449, ECK1443

Synonyms: yncB

Regulation Summary Diagram: ?

Subunit composition of NADPH-dependent curcumin/dihydrocurcumin reductase = [CurA]2
         NADPH-dependent curcumin/dihydrocurcumin reductase subunit = CurA

Summary:
The purified product of Escherichia coli gene curA was shown to catalyze the NADPH-dependent, sequential reduction of curcumin first to the stable intermediate dihydrocurcumin, and then to the final end product tetrahydrocurcumin. Curcumin is a plant secondary metabolite and curcumin metabolizing microorganisms were found in samples of human feces. The organism with the highest curcumin converting activity was isolated, cultured and identified as Escherichia coli. High curcumin converting activity was also identified in Escherichia coli K-12 substr. DH10B whose genome sequence has been determined. The curcumin metabolizing enzyme was purified from extracts of this organism and used to identify its encoding gene. Following gene cloning and overexpression, the recombinant enzyme was purified and characterized. The results showed that E. coli gene yncB encoded the curcumin-converting enzyme and it was renamed curA [Hassaninasab11].

CurA protein showed amino acid sequence similarity with some members of the medium-chain dehydrogenase/reductase (MDR) superfamily. Due to its non-requirement for Zn2+ the enzyme is a member of a zinc-independent family of the MDR superfamily. Creation of a C247A mutant in a putative NADPH binding site by site-directed mutagenesis and purification and assay of the resulting enzyme showed an approximately 50% reduction in activity in this mutant [Hassaninasab11].

The apparent molecular mass of the complex was determined by gel filtration chromatography and the apparent molecular mass of the subunit was determined by SDS-PAGE. The absorption spectrum of the enzyme suggested that the enzyme contained no bound cofactor. Qualitative and quantitative analysis for a variety of metals showed no metal content. The enzyme secondary structure showed a surprisingly wide range of pH stability from pH 4.5-12.0 as detrmined by far-UV circular dichroism spectra [Hassaninasab11].

Locations: cytosol

Map Position: [1,517,051 -> 1,518,088] (32.7 centisomes)
Length: 1038 bp / 345 aa

Molecular Weight of Polypeptide: 37.61 kD (from nucleotide sequence), 38.0 kD (experimental) [Hassaninasab11 ]

Molecular Weight of Multimer: 82.0 kD (experimental) [Hassaninasab11]

Unification Links: ASAP:ABE-0004834 , DIP:DIP-12748N , EchoBASE:EB3534 , EcoGene:EG13772 , EcoliWiki:b1449 , ModBase:P76113 , OU-Microarray:b1449 , PortEco:yncB , Protein Model Portal:P76113 , RefSeq:NP_415966 , RegulonDB:G6760 , SMR:P76113 , String:511145.b1449 , UniProt:P76113

Relationship Links: InterPro:IN-FAMILY:IPR002085 , InterPro:IN-FAMILY:IPR011032 , InterPro:IN-FAMILY:IPR013149 , InterPro:IN-FAMILY:IPR016040 , Panther:IN-FAMILY:PTHR11695 , Pfam:IN-FAMILY:PF00107

In Paralogous Gene Group: 103 (13 members)

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0055114 - oxidation-reduction process Inferred by computational analysis [UniProtGOA11a, GOA01a]
Molecular Function: GO:0042803 - protein homodimerization activity Inferred from experiment [Hassaninasab11]
GO:0008270 - zinc ion binding Inferred by computational analysis [GOA01a]
GO:0016491 - oxidoreductase activity Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0016628 - oxidoreductase activity, acting on the CH-CH group of donors, NAD or NADP as acceptor Inferred by computational analysis [GOA01]
Cellular Component: GO:0005829 - cytosol Inferred by computational analysis [DiazMejia09]

Gene Class: ORFs

Essentiality data for curA 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]

Credits:
Last-Curated ? 06-May-2008 by Keseler I , SRI International
Revised 18-Apr-2011 by Fulcher C , SRI International


Enzymatic reaction of: NADPH-dependent dihydrocurcumin reductase (NADPH-dependent curcumin/dihydrocurcumin reductase)

dihydrocurcumin + NADPH + H+ <=> tetrahydrocurcumin + NADP+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is favored in the direction shown.

In Pathways: curcumin degradation


Enzymatic reaction of: NADPH-dependent curcumin reductase (NADPH-dependent curcumin/dihydrocurcumin reductase)

curcumin + NADPH + H+ <=> dihydrocurcumin + NADP+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is favored in the direction shown.

Alternative Substrates for curcumin: trans-resveratrol [Hassaninasab11 ] , trans-2-octenal [Hassaninasab11 ] , 3-octen-2-one [Hassaninasab11 ] , 3-hepten-2-one [Hassaninasab11 ]

In Pathways: curcumin degradation

Summary:
Time course studies showed that the enzyme first reduces curcumin to dihydrocurcumin and then dihydrocurcumin is sequentially reduced to tetrahydrocurcumin. The reaction products dihydrocurcumin and tetrahydrocurcumin were identified by a combination of liquid chromatography electrospray ionization mass spectrometry, fast atom bombardment mass spectrometry and UV-visible absorption spectra. [Hassaninasab11].

The enzyme was shown to be NADPH-dependent. Activity loss during dialysis could be restored to high levels by addition of NADPH, while NADH was ineffective. It also had a relatively narrow substrate specificity, preferentially reducing curcumin. The alternative substrates 3-octene-2-one, 3-hepten-2-one, resveratrol and trans-2-octenal showed 59.7%, 28%, 14.8% and 8.13% of the activity towards curcumin, respectively [Hassaninasab11].

Metal analysis showed that the enzyme contained no metal ions and its activity was unaffected by addition of various metal ions, or chelating agents. The enzyme activity showed a surprisingly wide range of pH stability from pH 4.5-12.0. It was not inhibited by carbonyl-specific reagents, reducing reagents, or chelating agents [Hassaninasab11].

Inhibitors (Unknown Mechanism): diisopropyl fluorophosphate [Hassaninasab11] , iodoacetate [Hassaninasab11] , phenylmethanesulfonyl fluoride [Hassaninasab11] , 5,5'-dithio-bis-2-nitrobenzoate [Hassaninasab11] , N-ethylmaleimide [Hassaninasab11] , Hg2+ [Hassaninasab11] , Cu2+ [Hassaninasab11] , Fe2+ [Hassaninasab11] , Ag+ [Hassaninasab11] , Cd2+ [Hassaninasab11] , p-chloromercuribenzoate [Hassaninasab11]

Kinetic Parameters:

Substrate
Km (μM)
Citations
curcumin
29.0
[Hassaninasab11]

T(opt): 35 °C [Hassaninasab11]

pH(opt): 5.9 [Hassaninasab11]


Sequence Features

Feature Class Location Citations Comment
Amino-Acid-Sites-That-Bind 51
[UniProt12a]
UniProt: NADP; Non-Experimental Qualifier: by similarity.
Nucleotide-Phosphate-Binding-Region 160 -> 163
[UniProt11a]
UniProt: NADP; Non-Experimental Qualifier: by similarity.
Nucleotide-Phosphate-Binding-Region 182 -> 186
[UniProt12a]
UniProt: NADP; Non-Experimental Qualifier: by similarity.
Amino-Acid-Sites-That-Bind 186
[UniProt11a]
UniProt: NADP; Non-Experimental Qualifier: by similarity.
Amino-Acid-Sites-That-Bind 225
[UniProt11a]
UniProt: NADP; Non-Experimental Qualifier: by similarity.
Mutagenesis-Variant 247
[Hassaninasab11, UniProt12a]
Alternate sequence: C → A; UniProt: Exhibits a reduction of about 50% in activity, but is not inhibited by 5,5'-dithio-bis-2-nitrobenzoate.
Nucleotide-Phosphate-Binding-Region 247 -> 253
[UniProt11a]
UniProt: NADP; Non-Experimental Qualifier: by similarity.
Nucleotide-Phosphate-Binding-Region 282 -> 284
[UniProt11a]
UniProt: NADP; Non-Experimental Qualifier: by similarity.
Amino-Acid-Sites-That-Bind 333
[UniProt11a]
UniProt: NADP; Non-Experimental Qualifier: by similarity.


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
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 ].
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

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

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."

Hassaninasab11: Hassaninasab A, Hashimoto Y, Tomita-Yokotani K, Kobayashi M (2011). "Discovery of the curcumin metabolic pathway involving a unique enzyme in an intestinal microorganism." Proc Natl Acad Sci U S A 108(16):6615-20. PMID: 21467222

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

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

UniProt11a: UniProt Consortium (2011). "UniProt version 2011-11 released on 2011-11-22 00:00:00." Database.

UniProt12a: UniProt Consortium (2012). "UniProt version 2012-02 released on 2012-02-29 00:00:00." Database.

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."


Report Errors or Provide Feedback
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 Sat Nov 22, 2014, biocyc14.