Cloned (Comment) | Organism |
---|---|
gene fmoa, recombinant expression of His6-tagged wild-type and mutant isozymes ZvPNO in Escherichia coli strain BL21(DE3) | Zonocerus variegatus |
gene pno, recombinant expression of His6-tagged isozyme ZvPNO in Escherichia coli strain BL21(DE3) | Zonocerus variegatus |
Crystallization (Comment) | Organism |
---|---|
purified recombinant isozyme PA N-oxygenase (ZvFMOa), mixing of 6.5 mg/ml protein in 20 mM glycine-NaOH, pH 9.0, and 1 mM DTT with an equal amount of precipitant solution containing 20 mM bis-tris propane-NaOH, pH 7.5, 200 mM NaNO3, 22% w/v PEG 3000, 1 mM TMA-HCl, and 1 mM NADP+, equilibration against reservoir solution, 18°C, X-ray diffraction structure determiantion and analysis at 3.0 A resolution | Zonocerus variegatus |
purified recombinant isozyme PA N-oxygenase (ZvPNO) with bound FAD or in complex with FAD and NADP+, hanging drop vapour diffusion method, mixing of 5.6 mg/ml protein in 20 mM glycine-NaOH, pH 9.0, and 1 mM DTT with an equal amount of precipitant solution containing 20 mM Tris-HCl, pH 7.0, 200 mM MgCl2, and 15% w/v PEG 3350, with or without 1 mM NADP+, equilibration against reservoir solution, 18°C, X-ray diffraction structure determiantion and analysis at 1.6-1.89 A resolution | Zonocerus variegatus |
Protein Variants | Comment | Organism |
---|---|---|
F307Y | site-directed mutagenesis, the ZvFMOa F307Y mutation results in a decline in specific activity by a factor of 0.81 in comparison to wild-type ZvFMOa | Zonocerus variegatus |
F307Y/Y356A | site-directed mutagenesis, the ZvFMOa F307Y/Y356A double mutation results in an increase in specific activity by a factor of 1.51 in comparison to wild-type ZvFMOa | Zonocerus variegatus |
F354V | site-directed mutagenesis, the ZvFMOa F354V mutation results in an increase in specific activity by a factor of 2.75 in comparison to wild-type ZvFMOa | Zonocerus variegatus |
F354V/Y356A | site-directed mutagenesis, the ZvFMOa F354V/Y356A double mutation results in an increase in specific activity by a factor of 1.5 in comparison to wild-type ZvFMOa | Zonocerus variegatus |
additional information | the beneficial effect of a more accessible substrate entry path might possibly be partly antagonized by a loss of substrate affinity when both bulky aromatic amino acids are exchanged for smaller substituents. The presence of proline within the ZvFMOa helix does not interfere with its potential role in substrate turnover | Zonocerus variegatus |
P388S | site-directed mutagenesis, the specific activity of this variant does not differ significantly from that of wild-type ZvFMOa | Zonocerus variegatus |
Y356A | site-directed mutagenesis, the ZvFMOa Y356A mutation results in an increase in specific activity by a factor of 2.45 in comparison to wild-type ZvFMOa | Zonocerus variegatus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
senecionine + NADPH + H+ + O2 | Zonocerus variegatus | - |
senecionine N-oxide + NADP+ + H2O | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Zonocerus variegatus | L0N8F3 | - |
- |
Zonocerus variegatus | L0N8S9 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged isozyme ZvPNO from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration | Zonocerus variegatus |
recombinant His6-tagged wild-type and mutant isozymes ZvFMOa from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration | Zonocerus variegatus |
Source Tissue | Comment | Organism | Textmining |
---|---|---|---|
hemolymph | - |
Zonocerus variegatus | - |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | class B FMOs such as those from Zonocerus variegatus consist of two domains, each of which contains a dinucleotide-binding domain in the form of a Rossmann fold, which is responsible for cofactor binding. In its native state, oxidized FAD is tightly bound to the enzyme as a prosthetic group. NADPH is recruited as a co-substrate and transfers reducing equivalents to FAD. Upon the reaction of reduced FAD by molecular oxygen, a C4a-hydroperoxy-FAD intermediate is formed. This intermediate is capable of inserting one O atom into a substrate compound and ends up as C4a-hydroxy-FAD, which may then release the second O atom as part of a water molecule, restoring the oxidized FAD cofactor. A cocked-gun mechanism has been proposed for the reaction cycle of FMOs, meaning that the activated C4a-hydroperoxy-FAD intermediate can be stabilized within the enzyme until a substrate accesses the active site and is then immediately oxygenated. The presence of NADP+ seems to be crucial for the stabilization of this intermediate and therefore it has to remain bound to the enzyme throughout the whole catalytic cycle, making it the last compound to be released from the enzyme. NADP+ binding structure analysis, overview. The NADP+ cofactor is bound to the GXGXXG motif (residues 191-196) of the smaller structural domain in an extended conformation via hydrogen bonds to its diphosphate moiety. The 2'-phosphate of NADP+ is coordinated by Lys223 and His351. Interactions between the nicotinamide moiety and Phe64 and Arg398 as well as between the ribose moiety and Asn66 further support the binding and positioning of the cofactor. In comparison to the ZvPNO-FAD complex, the presence of NADP+ leads to a small conformational change of helix alpha4 such that it provides additional cofactor stabilization by interactions between a positive partial charge of the helix dipole and the diphosphate of NADP+. The conformational change in the preceding loop region is caused by a flipping alanine, which reduces steric hindrance when binding the NADP+ cofactor. The ribose moiety of NADP+ and Asn66 are supposed to coordinate molecular oxygen and stabilize the C4a-hydroperoxy-FAD intermediate | Zonocerus variegatus | ? | - |
- |
|
additional information | class B FMOs such as those from Zonocerus variegatus consist of two domains, each of which contains a dinucleotide-binding domain in the form of a Rossmann fold, which is responsible for cofactor binding. In its native state, oxidized FAD is tightly bound to the enzyme as a prosthetic group. NADPH is recruited as a co-substrate and transfers reducing equivalents to FAD. Upon the reaction of reduced FAD by molecular oxygen, a C4a-hydroperoxy-FAD intermediate is formed. This intermediate is capable of inserting one O atom into a substrate compound and ends up as C4a-hydroxy-FAD, which may then release the second O atom as part of a water molecule, restoring the oxidized FAD cofactor. A cocked-gun mechanism has been proposed for the reaction cycle of FMOs, meaning that the activated C4a-hydroperoxy-FAD intermediate can be stabilized within the enzyme until a substrate accesses the active site and is then immediately oxygenated. The presence of NADP+ seems to be crucial for the stabilization of this intermediate and therefore it has to remain bound to the enzyme throughout the whole catalytic cycle, making it the last compound to be released from the enzyme. The ribose moiety of NADP+ and Asn66 are supposed to coordinate molecular oxygen and stabilize the C4a-hydroperoxy-FAD intermediate | Zonocerus variegatus | ? | - |
- |
|
senecionine + NADPH + H+ + O2 | - |
Zonocerus variegatus | senecionine N-oxide + NADP+ + H2O | - |
? |
Subunits | Comment | Organism |
---|---|---|
homodimer | HPLC-based size-exclusion chromatography (SEC) with refractive-index (RI) and multi-angle laser light-scattering (MALS) detectors, the isozyme ZvPNO shows a unique dimeric arrangement as well as small conformational changes within the active site. A unique flexible helix close to the active site is observed which is likely to be involved in substrate binding and/or product release. Symmetrical homodimers with twofold symmetry can be observed within the ZvPNO crystal structure with a distinct type of orientation | Zonocerus variegatus |
More | FMO isozymes' structure analysis and comparisons, overview | Zonocerus variegatus |
Synonyms | Comment | Organism |
---|---|---|
flavin-dependent monooxygenase | - |
Zonocerus variegatus |
fmoa | - |
Zonocerus variegatus |
PA N-oxygenase | - |
Zonocerus variegatus |
PNO | - |
Zonocerus variegatus |
pyrrolizidine alkaloid N-oxygenase | - |
Zonocerus variegatus |
ZvFMOa | - |
Zonocerus variegatus |
ZvPNO | - |
Zonocerus variegatus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Zonocerus variegatus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
9 | - |
assay at | Zonocerus variegatus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | - |
Zonocerus variegatus | |
FAD | binding structure analysis, overview | Zonocerus variegatus | |
NADPH | - |
Zonocerus variegatus |
General Information | Comment | Organism |
---|---|---|
evolution | during evolution, different insect lineages have developed specialized FMOs, pyrrolizidine alkaloid N-oxygenases (PNOs), as a counterstrategy to cope with pyrrolizidine alkaloids (PAs), which are toxic compounds that are produced by certain angiosperm species as part of their chemical defence against herbivores. PAs are produced by plants in their nontoxic polar N-oxide form. Isonzyme ZvPNO belongs to the flavin-dependent monooxygenases (FMOs), it is a member of class B (of six different FMO subclasses, A-F) within the family of flavin-dependent monooxygenases that originates from a more highly developed organism than yeast. Despite the differences in sequence between family members, their overall structure is very similar. PA N-oxygenases are the only functionally characterized FMOs found to date in insects | Zonocerus variegatus |
additional information | FMO isozymes' structure analysis and comparisons, overview. Based on initial docking experiments with the crystal structure of ZvPNO and different PA substrates, Tyr307 is identified as the most promising part of the binding pocket, potentially forming hydrogen bonds to a variety of PA substrates. Additionally, when comparing the crystal structures of ZvPNO with a homology model of ZvFMOa, a tyrosine residue (Tyr356) blocking the substrate entrance in the latter model is observed | Zonocerus variegatus |
physiological function | Zonocerus variegatus expresses three flavin-dependent monooxygenase (ZvFMO) isoforms, ZvFMOa, ZvFMOc and ZvPNO, which contribute to a counterstrategy against pyrrolizidine alkaloids (PAs). PAs are protoxic compounds produced by some angiosperm lineages as a chemical defence against herbivores. While ZvPNO is the most potent isoform, ZvFMOa and ZvFMOc show a specific activity that is 8-300fold lower, depending on the PA substrate. N-Oxygenation of PAs and the accumulation of PA N-oxides within their haemolymph result in two evolutionary advantages for these insects: (1) they circumvent the defence mechanism of their food plants and (2) they can use PA N-oxides to protect themselves against predators, which cannot cope with the toxic PAs. Despite a high degree of sequence identity and a similar substrate spectrum, the three ZvFMO isoforms differ greatly in enzyme activity. PA N-oxygenase (ZvPNO) is the most active ZvFMO isoform | Zonocerus variegatus |
physiological function | Zonocerus variegatus expresses three flavin-dependent monooxygenase (ZvFMO) isoforms, ZvFMOa, ZvFMOc and ZvPNO, which contribute to a counterstrategy against pyrrolizidine alkaloids (PAs). PAs are protoxic compounds produced by some angiosperm lineages as a chemical defence against herbivores. While ZvPNO is the most potent isoform, ZvFMOa and ZvFMOc show a specific activity that is 8-300fold lower, depending on the PA substrate. N-Oxygenation of PAs and the accumulation of PA N-oxides within their hemolymph result in two evolutionary advantages for these insects: (1) they circumvent the defence mechanism of their food plants and (2) they can use PA N-oxides to protect themselves against predators, which cannot cope with the toxic PAs. Despite a high degree of sequence identity and a similar substrate spectrum, the three ZvFMO isoforms differ greatly in enzyme activity. PA N-oxygenase (ZvPNO) is the most active ZvFMO isoform | Zonocerus variegatus |