Literature summary for 1.14.13.101 extracted from

Kubitza, C.; Faust, A.; Gutt, M.; Gaeth, L.; Ober, D.; Scheidig, A.J.
Crystal structure of pyrrolizidine alkaloid N-oxygenase from the grasshopper Zonocerus variegatus (2018), Acta Crystallogr. Sect. D, 74, 422-432 .

Search for more literature for 1.14.13.101

Cloned(Commentary)

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 (Commentary)

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

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

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/ Products (Substrates)

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

Organism	UniProt	Comment	Textmining
Zonocerus variegatus	L0N8F3	-	-
Zonocerus variegatus	L0N8S9	-	-

Purification (Commentary)

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

Source Tissue	Comment	Organism	Textmining
hemolymph	-	Zonocerus variegatus	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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

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

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 [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Zonocerus variegatus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
9	-	assay at	Zonocerus variegatus

Cofactor

Cofactor	Comment	Organism
FAD	-	Zonocerus variegatus
FAD	binding structure analysis, overview	Zonocerus variegatus
NADPH	-	Zonocerus variegatus

General Information

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