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Literature summary extracted from

  • Liu, S.; Su, T.; Zhang, C.; Zhang, W.M.; Zhu, D.; Su, J.; Wei, T.; Wang, K.; Huang, Y.; Guo, L.; Xu, S.; Zhou, N.Y.; Gu, L.
    Crystal structure of PnpCD, a two-subunit hydroquinone 1,2-dioxygenase, reveals a novel structural class of Fe2+-dependent dioxygenases (2015), J. Biol. Chem., 290, 24547-24560 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.13.11.66 genes pnpCD, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) Pseudomonas sp.

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.13.11.66 purified full-length or proteolytically truncated PnpCD in apo form and in complex with Fe3+ or substrate analogue hydroxybenzonitrile and Cd2+, i.e. apo-PnpCD, PnpCD-Fe3+, and PnpCD-Cd2+-HBN, sitting-drop vapor diffusion method, 15-20 mg/ml protein in 10mM Tris-HCl, pH 8.0, and 100 mM NaCl, is mixed with reservoir solution containing 0.2 M sodium thiocyanate, 20% w/v PEG 3350, 20°C, method optimization, X-ray diffraction structure determination and analysis Pseudomonas sp.

Protein Variants

EC Number Protein Variants Comment Organism
1.13.11.66 E248Q site-directed mutagenesis, the mutation results in complete loss of enzyme activity Pseudomonas sp.
1.13.11.66 F264A site-directed mutagenesis, the mutation results in almost complete loss of enzyme activity Pseudomonas sp.
1.13.11.66 F79A site-directed mutagenesis, the mutation results in almost complete loss of enzyme activity Pseudomonas sp.
1.13.11.66 L252A site-directed mutagenesis, the mutation results in a 70% loss of enzyme activity Pseudomonas sp.
1.13.11.66 L313A site-directed mutagenesis, the mutation results in almost complete loss of enzyme activity Pseudomonas sp.
1.13.11.66 V315A site-directed mutagenesis, the mutation results in a 50% loss of enzyme activity Pseudomonas sp.
1.13.11.66 W230A site-directed mutagenesis, the mutation results in a 70% loss of enzyme activity Pseudomonas sp.
1.13.11.66 W273a site-directed mutagenesis, the mutation results in almost complete loss of enzyme activity Pseudomonas sp.
1.13.11.66 W76A site-directed mutagenesis, the mutation results in almost complete loss of enzyme activity Pseudomonas sp.

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.13.11.66 Fe2+ a Fe2+-dependent dioxygenase, selectively utilizes Fe2+for its catalytic reaction. Four residues of enzyme PnpD, His256, Asn258, Glu262, and His303, coordinate the iron ion Pseudomonas sp.
1.13.11.66 Fe3+ binding structure, overview Pseudomonas sp.
1.13.11.66 additional information Fe3+, Mn2+, Co2+, Ni2+, Cu2+, and Cd2+ cannot substitute for Fe2+ Pseudomonas sp.

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.13.11.66 4-nitrophenol + O2 Pseudomonas sp.
-
 ?
-
 ?
1.13.11.66 benzene-1,4-diol + O2 Pseudomonas sp.
-
 (2Z,4E)-4-hydroxy-6-oxohexa-2,4-dienoate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.13.11.66 Pseudomonas sp. C1I210 AND C1I209 beta- and alpha-subunit
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.13.11.66 recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration Pseudomonas sp.

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.13.11.66 4-nitrophenol + O2
-
 Pseudomonas sp. ?
-
 ?
1.13.11.66 benzene-1,4-diol + O2
-
 Pseudomonas sp. (2Z,4E)-4-hydroxy-6-oxohexa-2,4-dienoate
-
 ?

Subunits

EC Number Subunits Comment Organism
1.13.11.66 heterotetramer 2 * 38300, subunit alpha, + 2 * 18000, subunit beta Pseudomonas sp.
1.13.11.66 More the PnpCD structure contains a pseudo cupin and an iron metallocenter in the catalytic PnpD. Both the PnpC and the C-terminal domains of PnpD comprise a conserved cupin fold, whereas PnpC cannot form a competent metal binding pocket as can PnpD cupin, structure analysis, overview Pseudomonas sp.

Synonyms

EC Number Synonyms Comment Organism
1.13.11.66 PnpCD
-
 Pseudomonas sp.
1.13.11.66 two-subunit hydroquinone 1,2-dioxygenase
-
 Pseudomonas sp.

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.13.11.66 25
-
 assay at Pseudomonas sp.

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.13.11.66 7
-
 assay at Pseudomonas sp.

General Information

EC Number General Information Comment Organism
1.13.11.66 metabolism the two-subunit hydroquinone 1,2-dioxygenase PnpCD is the ring cleavage enzyme in para-nitrophenol catabolism Pseudomonas sp.
1.13.11.66 additional information the PnpCD structure contains a pseudo cupin and a iron metallocenter in the catalytic PnpD, which adds to understanding of the ring cleavage mechanism of dioxygenases, structure analysis, overview Pseudomonas sp.
1.13.11.66 physiological function hydroquinone 1,2-dioxygenase PnpCD is the key enzyme in the hydroquinone pathway of para-nitrophenol degradation, catalyzes the ring cleavage of hydroquinone to gamma-hydroxymuconic semialdehyde Pseudomonas sp.