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Literature summary extracted from

  • Fiorillo, A.; Federico, R.; Polticelli, F.; Boffi, A.; Mazzei, F.; Di Fusco, M.; Ilari, A.; Tavladoraki, P.
    The structure of maize polyamine oxidase K300M mutant in complex with the natural substrates provides a snapshot of the catalytic mechanism of polyamine oxidation (2011), FEBS J., 278, 809-821.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.5.3.14 recombinant expression of His-tagged wild-type and mutant ZmPAO enzymes in Pichia pastoris strain X-33 Zea mays

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.5.3.14 purified recombinant mutant ZmPAO-K300M, hanging drop vapor diffusion method, mixing of 9.0 mg/ml protein in 50 mM sodium acetate, pH 5.5, with an equal volume of reservoir solution containing ammonium sulfate in a concentration range 2.2–2.8 m and 100 mm sodium acetate, pH 4.6, 20°C, two weeks, X-ray diffraction structure determination and analysis at 2.9 A resolution,mlecular displacement, modelling Zea mays

Protein Variants

EC Number Protein Variants Comment Organism
1.5.3.14 K300M site-directed mutagenesis, catalyitically impaired active site mutant, that is less glycosylated than the wild-type enzyme. The mutant shows a 1400fold decrease in the rate of flavin reduction. Substrates are bound in an out-of-register mode and the HOH309 water molecule is absent in the enzyme–substrate complexes. K300 mutation brings about a 60 mV decrease in the FAD redox potential and a 30fold decrease in the FAD reoxidation rate, within a virtually unaltered geometry of the catalytic pocket Zea mays

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.5.3.14 additional information Zea mays the enzyme from Zea mays oxidizes the carbon on the endo-side of the N5-nitrogen of spermidine and spermine ?
-
 ?
1.5.3.14 spermidine + O2 + H2O Zea mays
-
 propane-1,3-diamine + 4-aminobutanal + H2O2
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.5.3.14 Zea mays
-
-
-

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
1.5.3.14 glycoprotein the wild-type enzyme's glycosylation site is at residue N77. Modelling of wild-type subunit C with a branched chain of five ordered sugars (two N-acetyl-D-glucosamine residues as well as a fucose and two mannose residues). The ZmPAO-K300M mutant appears to be less glycosylated than the wild-type enzyme Zea mays

Purification (Commentary)

EC Number Purification (Comment) Organism
1.5.3.14 recombinant His-tagged wild-type and mutant ZmPAO enzymes from Pichia pastoris strain X-33 bx nickel affinity chromatography Zea mays

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.5.3.14 additional information the enzyme from Zea mays oxidizes the carbon on the endo-side of the N5-nitrogen of spermidine and spermine Zea mays ?
-
 ?
1.5.3.14 spermidine + O2 + H2O
-
 Zea mays propane-1,3-diamine + 4-aminobutanal + H2O2
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.5.3.14 PAO
-
 Zea mays

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.5.3.14 25
-
 assay at Zea mays

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.5.3.14 4
-
 assay at Zea mays

Cofactor

EC Number Cofactor Comment Organism Structure
1.5.3.14 FAD dependent on. Residue K300 is hydrogen-bound to the N5 atom of FAD via a H2O molecule (HOH309), the only solvent molecule present inside the catalytic site in the enzyme inhibitor complexes. In turn, K300 is the only active-site residue whose conformation changes upon FAD reduction, possibly allowing for a reorientation of the HOH309 molecule. The water molecule might thus function as a hydrogen-bond acceptor with the protonated N5 atom of reduced FAD Zea mays

General Information

EC Number General Information Comment Organism
1.5.3.14 malfunction the ZmPAO-K300M mutant is catalytically impaired with a 1400fold decrease in the rate of flavin reduction. Substrates are bound in an out-of-register mode and the HOH309 water molecule is absent in the enzyme-substrate complexes. K300 mutation brings about a 60 mV decrease in the FAD redox potential and a 30fold decrease in the FAD reoxidation rate, within a virtually unaltered geometry of the catalytic pocket Zea mays
1.5.3.14 additional information active site structure of wild-type and mutant K300M enzymes, overview. The active site is formed by a catalytic tunnel in which the N5 atom of FAD lies in close proximity to the K300 side chain, the only active-site residue conserved in all PAOs. A water molecule, HOH309, is hydrogen-bound to the amino group of K300. The HOH309-K300 couple plays a major role in multiple steps of ZmPAO catalytic mechanism, such as correct substrate binding geometry as well as FAD reduction and reoxidation kinetics. Substrate binding mechanism and structure, and comparison to Saccharomyces cerevisiae Fms1, EC 1.5.3.17, overview. The differences include a planar conformation of the isoalloxazine ring in Fms1 versus a highly bent conformation in ZmPAO and important substitutions in the relevant topological positions of the active site, i.e. E170W, F171H, E62H, V196N, S87D, F318K, F403Y, V331F, T348L, Y169L and Y298L, numbering referring to ZmPAO. The substrate-binding site of Fms1 is more hydrophobic than that of ZmPAO Zea mays