Cloned (Comment) | Organism |
---|---|
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta(DE3)/pLysS | Rhodococcus opacus |
Crystallization (Comment) | Organism |
---|---|
- |
Rhodococcus opacus |
purified recombinant His-tagged wild-type enzyme, sitting drop vapor diffusion method, mixing equal volumes of protein solution and reservoir solution. The reservoir solution contains 0.1 M Tris, pH 7.25, 1.5 M (NH4)2SO4, and 4% v/v glycerol, diffraction-quality crystals of PnpA1 appear within 5 days at 20°C, X-ray diffraction structure determination and analysis at 2.5 A resolution | Rhodococcus opacus |
Protein Variants | Comment | Organism |
---|---|---|
H293A | complete loss of activity | Rhodococcus opacus |
H293A | site-directed mutagenesis, inactive mutant | Rhodococcus opacus |
M192I | site-directed mutagenesis, the mutant shows significantly reduced activity compared to wild-type enzyme | Rhodococcus opacus |
N450A | site-directed mutagenesis, the mutant shows slightly altered substrate specificity compared to wild-type enzyme | Rhodococcus opacus |
N450A | variant displays improved activity for 4-nitrocatechol and 2-chloro-4-nitrophenol | Rhodococcus opacus |
R100A | complete loss of activity | Rhodococcus opacus |
R100A | site-directed mutagenesis, inactive mutant | Rhodococcus opacus |
R370A | complete loss of activity | Rhodococcus opacus |
R370A | site-directed mutagenesis, inactive mutant | Rhodococcus opacus |
V292A | site-directed mutagenesis, the mutant shows significantly reduced activity compared to wild-type enzyme | Rhodococcus opacus |
V292L | site-directed mutagenesis, the mutant shows slightly reduced activity compared to wild-type enzyme | Rhodococcus opacus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
4-nitrophenol + NADH + H+ + O2 | Rhodococcus opacus | - |
4-nitrocatechol + NAD+ + H2O | - |
? | |
4-nitrophenol + NADH + H+ + O2 | Rhodococcus opacus JCM 13270 | - |
4-nitrocatechol + NAD+ + H2O | - |
? | |
4-nitrophenol + NADH + H+ + O2 | Rhodococcus opacus RKJ300 | - |
4-nitrocatechol + NAD+ + H2O | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Rhodococcus opacus | - |
- |
- |
Rhodococcus opacus JCM 13270 | - |
- |
- |
Rhodococcus opacus RKJ300 | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta(DE3)/pLysS by nickel affinity chromatography, gel filtration, and ultrafiltration | Rhodococcus opacus |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
4-nitrophenol + NADH + H+ + O2 = 4-nitrocatechol + NAD+ + H2O | proposed catalytic mechanism of PNP 2-monooxygenase, PnpA1, overview | Rhodococcus opacus |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.0128 | - |
substrate 2-chloro-4-nitrophenol, pH 7.6, 22°C | Rhodococcus opacus |
0.0179 | - |
substrate 4-nitrocatechol, pH 7.6, 22°C | Rhodococcus opacus |
0.034 | - |
substrate 4-nitrophenol, pH 7.6, 22°C | Rhodococcus opacus |
12.8 | - |
substrate 2-chloro-4-nitrophenol, pH 7.6, 23°C | Rhodococcus opacus |
17.9 | - |
substrate 4-nitrocatechol, pH 7.6, 23°C | Rhodococcus opacus |
34.2 | - |
substrate 4-nitrophenol, pH 7.6, 23°C | Rhodococcus opacus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-chloro-4-nitrophenol + NADH + H+ + O2 | - |
Rhodococcus opacus | 2-chloro-4-nitrocatechol + NAD+ + H2O | - |
? | |
2-chloro-4-nitrophenol + NADH + H+ + O2 | - |
Rhodococcus opacus JCM 13270 | 2-chloro-4-nitrocatechol + NAD+ + H2O | - |
? | |
2-chloro-4-nitrophenol + NADH + H+ + O2 | - |
Rhodococcus opacus RKJ300 | 2-chloro-4-nitrocatechol + NAD+ + H2O | - |
? | |
2-chloro-4-nitrophenol + NADPH + O2 | - |
Rhodococcus opacus | 1,2,4-trihydroxybenzene + ? | - |
? | |
2-chloro-4-nitrophenol + NADPH + O2 | - |
Rhodococcus opacus JCM 13270 | 1,2,4-trihydroxybenzene + ? | - |
? | |
2-chloro-4-nitrophenol + NADPH + O2 | - |
Rhodococcus opacus RKJ300 | 1,2,4-trihydroxybenzene + ? | - |
? | |
4-nitrocatechol + NADH + H+ + O2 | - |
Rhodococcus opacus | 4-nitrobenzene-1,2,3-triol + NAD+ + H2O | - |
? | |
4-nitrocatechol + NADH + H+ + O2 | - |
Rhodococcus opacus JCM 13270 | 4-nitrobenzene-1,2,3-triol + NAD+ + H2O | - |
? | |
4-nitrocatechol + NADH + H+ + O2 | - |
Rhodococcus opacus RKJ300 | 4-nitrobenzene-1,2,3-triol + NAD+ + H2O | - |
? | |
4-nitrocatechol + NADPH + H+ + O2 | - |
Rhodococcus opacus | 4-nitrobenzene-1,2,3-triol + NADP+ + H2O | - |
? | |
4-nitrophenol + NADH + H+ + O2 | - |
Rhodococcus opacus | 4-nitrocatechol + NAD+ + H2O | - |
? | |
4-nitrophenol + NADH + H+ + O2 | - |
Rhodococcus opacus JCM 13270 | 4-nitrocatechol + NAD+ + H2O | - |
? | |
4-nitrophenol + NADH + H+ + O2 | - |
Rhodococcus opacus RKJ300 | 4-nitrocatechol + NAD+ + H2O | - |
? | |
4-nitrophenol + NADPH + H+ + O2 | - |
Rhodococcus opacus | 4-nitrocatechol + NADP+ + H2O | - |
? | |
additional information | PnpA1 is the oxygenase component of the two-component PNP 2-monooxygenase from Gram-positive Rhodococcus imtechensis strain RKJ300. It also catalyzes the hydroxylation of 4-nitrocatechol (4NC) and 2-chloro-4-nitrophenol (2C4NP) | Rhodococcus opacus | ? | - |
- |
|
additional information | PnpA1 is the oxygenase component of the two-component PNP 2-monooxygenase from Gram-positive Rhodococcus imtechensis strain RKJ300. It also catalyzes the hydroxylation of 4-nitrocatechol (4NC) and 2-chloro-4-nitrophenol (2C4NP) | Rhodococcus opacus JCM 13270 | ? | - |
- |
|
additional information | PnpA1 is the oxygenase component of the two-component PNP 2-monooxygenase from Gram-positive Rhodococcus imtechensis strain RKJ300. It also catalyzes the hydroxylation of 4-nitrocatechol (4NC) and 2-chloro-4-nitrophenol (2C4NP) | Rhodococcus opacus RKJ300 | ? | - |
- |
Subunits | Comment | Organism |
---|---|---|
homotetramer | crystal structure analysis, the functional unit of PnpA1 is a tetramer, overview | Rhodococcus opacus |
More | the PnpA1 monomer consists of 15 alpha-helices, 13 beta-strands, and four 310 helices and can be divided into three sequential segments based on their constituent secondary structural elements. Those three segments are tightly interconnected in the three-dimensional structure. Moreover, they form three faces of the hydrophobic substrate pocket. The first segment (residues 1 to 147) is composed of six helices (alpha1 to alpha6) and three short beta-strands (beta1 to beta3). This area is located in the periphery of the tetramer and constitutes the inner side of the pocket. The second segment (residues 148 to 277) has eight beta-strands (b4 to b11). These beta-strands are arranged as a small beta-barrel. The third segment, namely, the C-terminal segment, is composed of 8 helices (alpha7 to alpha15) and 2 short beta-strands (beta12 and beta13). Six alpha-helices (alpha7, alpha8, alpha9, alpha11, alpha12, and alpha13) form a helix bundle, which is mainly maintained by hydrophobic interaction among them. This segment is located at the tetramerization interface and involved in interacting with neighboring subunits. Alpha14 and alpha15 are the most extensive two of the eight helices. They clearly protrude from the protein core and extend into the neighboring subunit. No electron density is observed for residues 162 to 168 and residues 506 to 528 because of their high flexibility, which is common among PnpA1's homologues | Rhodococcus opacus |
tetramer | crystallization data | Rhodococcus opacus |
Synonyms | Comment | Organism |
---|---|---|
PNP 2-monooxygenase | - |
Rhodococcus opacus |
PnpA1 | - |
Rhodococcus opacus |
PnpA1 | oxygenase component | Rhodococcus opacus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
22 | - |
assay at room temperature | Rhodococcus opacus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.6 | - |
assay at | Rhodococcus opacus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | FAD binding site structure of PnpA1, molecular docking of FAD, overview | Rhodococcus opacus | |
FAD | the si-side of FAD is exposed to solvent, and the re-side faces the substrate-binding site. The loop from positions 449 to 454, loop 187-201, helices alpha13, alpha14, and loop 154-175 are responsible for coordinating FAD. The former three interact with the isoalloxazine ring of FAD, while the latter two interact with the AMP group | Rhodococcus opacus | |
NADPH | - |
Rhodococcus opacus |
General Information | Comment | Organism |
---|---|---|
evolution | PnpA1 is structurally determined to be a member of the group D flavin-dependent monooxygenases with an acyl coenzyme A (acyl-CoA) dehydrogenase fold, crystal structure analysis, overview. PnpA1 shows an obvious difference in substrate selectivity with its close homologues TcpA and TftD, which may be caused by the unique Thr296 and a different conformation in the loop from positions 449 to 454 (loop 449-454) | Rhodococcus opacus |
malfunction | an N450A variant is found with improved activity for 4NC and 2C4NP, probably because of reduced steric hindrance | Rhodococcus opacus |
metabolism | para-nitrophenol (PNP) is a hydrolytic product of organophosphate insecticides, such as parathion and methylparathion, in soil. Aerobic microbial degradation of PNP has been classically shown to proceed via the benzenetriol (BT) pathway in Gram-positive degraders. The BT pathway is initiated by a two-component PNP 2-monooxygenase. Comparison of the degradation pathways in Gram-negative and Gram-positive strains. Degradation pathways of PNP and 4NC in Gram-negative strains. PNP and 4NC are oxidized to p-benzoquinone and hydroxyl-1,4-benzoquinone, respectively, and the latter two can be reduced and degraded by other enzymes. Degradation pathways of PNP and 2C4NP in Gram-positive strains. PnpA1 oxidizes PNP to 4NC, and the latter is further oxidized to hydroxyl-1,4-benzoquinone, which can be nonenzymatically reduced to BT. 2C4NP can also be oxidized to produce chloro-1,4-benzoquinone, which is immediately converted to hydroxyl-1,4-benzoquinone by hydrolytic dechlorination and reduced to BT | Rhodococcus opacus |
metabolism | PnpA1 is a member of the group D flavin-dependent monooxygenases with an acyl-CoA dehydrogenase fold. Residues Arg100 and His293 are directly involved in catalysis. The bulky side chain of Val292 pushes the substrate toward FAD, hence positioning the substrate properly | Rhodococcus opacus |
additional information | PnpA1 is the oxygenase component of the two-component PNP 2-monooxygenase from Gram-positive Rhodococcus imtechensis strain RKJ300. It also catalyzes the hydroxylation of 4-nitrocatechol (4NC) and 2-chloro-4-nitrophenol (2C4NP). The crystal structure and site-directed mutagenesis underlined the direct involvement of residues Arg100 and His293 in catalysis. The bulky side chain of residue Val292 is proposed to push the substrate toward flavin adenine dinucleotide (FAD), hence positioning the substrate properly. Different PNP binding manners determine the choice of ortho- or para-hydroxylation on PNP by single-component PNP 4-monooxygenases and two-component PNP 2-monooxygenases. Substrate binding site structure of PnpA1, overview. For two-component flavin-dependent monooxygenases, reduced flavin must be bound to the enzyme before the substrate's entry. The PnpA1-FAD model shows that a substrate can only enter the binding pocket through a narrow hydrophobic tunnel, mainly consisting of Val156, Leu456, and Leu207, because of the prepositioned FAD. The perimeter of the substrate-binding site is mainly made with four fragments including a7, loop 449-454, loop 93-102, and loop 154-175. Among those fragments, loop 449-454 has a relatively high temperature factor and shows the most heterogeneous conformations among the four subunits, reflecting its flexibility. The residues lining the substrate-binding pocket are predominantly hydrophobic ones, including Phe446, Phe155, Phe289, Val292, and Leu207. Molecular docking of FAD and substrates | Rhodococcus opacus |
physiological function | PnpA1 is the oxygenase component of the two-component PNP 2-monooxygenase from Gram-positive Rhodococcus imtechensis strain RKJ300. It also catalyzes the hydroxylation of 4-nitrocatechol (4NC) and 2-chloro-4-nitrophenol (2C4NP) | Rhodococcus opacus |