Literature summary for 1.13.11.66 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 .

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Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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.

Crystallization (Comment)

Organism

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

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

Metals/Ions

Metals/Ions	Comment	Organism
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.
Fe3+	binding structure, overview	Pseudomonas sp.
additional information	Fe3+, Mn2+, Co2+, Ni2+, Cu2+, and Cd2+ cannot substitute for Fe2+	Pseudomonas sp.

Natural Substrates/ Products (Substrates)

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

Organism

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

Purification (Commentary)

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

Substrates and Products (Substrate)

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

Subunits

Subunits	Comment	Organism
heterotetramer	2 * 38300, subunit alpha, + 2 * 18000, subunit beta	Pseudomonas sp.
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

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

Temperature Optimum [°C]

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

pH Optimum

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

General Information

General Information	Comment	Organism
metabolism	the two-subunit hydroquinone 1,2-dioxygenase PnpCD is the ring cleavage enzyme in para-nitrophenol catabolism	Pseudomonas sp.
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.
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.