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Literature summary extracted from

  • Ohta, S.; Ishida, Y.; Usami, S.
    High-level expression of cold-tolerant pyruvate, orthophosphate dikinase from a genomic clone with site-directed mutations in transgenic maize (2006), Mol. Breed., 18, 29-38.
    View publication on PubMed

Activating Compound

EC Number Activating Compound Comment Organism Structure
2.7.9.1 additional information like the native PPDK of maize the activity of the cold-tolerant recombinant enzyme construct is regulated by the PPDK regulatory protein RP Zea mays

Application

EC Number Application Comment Organism
2.7.9.1 agriculture to improve the cold stability of the PPDK enzyme, studies on cold tolerance in plants Zea mays

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.9.1 Escherichia coli, three constructs, one construct consisting of the 3'-part of Flaveria brownii (Asteraceae) of cold tolerant PPDK fused to maize PPDK (15th exon), another construct includes a set of point mutations to substitute all of the 17 residues that differ between the 3'-parts of maize and Flaveria brownii PPDK, respectively, the whole genomic sequence of the maize PPDK gene is included as a control, transformation of constructs into maize inbred line A188 by Agrobacterium tumefaciens (strain LBA4404), individual range of variation in the amount of PPDK among regenerated plants, crude leaf extracts of some transformed plants produce a large amount of cold tolerant recombinant enzyme and reveal a greatly improved cold tolerance especially by using the construct altered at 17 amino acid positions Zea mays

Protein Variants

EC Number Protein Variants Comment Organism
2.7.9.1 additional information point mutations introduced into exons 15, 16, 17, 18, and 19 of the maize PPDK gene to generate a construct altered at 17 amino acid positions at the C-terminus that mimicks the 3'-part of PPDK of Flaveria brownii (Asteraceae) encoding the C-terminal part of PPDK responsible for cold tolerance, two other constructs represent a fusion protein of the 3'-part of PPDK of Flaveria brownii (Asteraceae) and maize PPDK, a further construct of maize PPDK serves as a control Zea mays

Organism

EC Number Organism UniProt Comment Textmining
2.7.9.1 Zea mays P11155
-
-

Source Tissue

EC Number Source Tissue Comment Organism Textmining
2.7.9.1 leaf maize inbred line A188, recombinant cold tolerant PPDK protein, 3'-part of Flaveria brownii (Asteraceae) PPDK fused to maize PPDK, exon-intron boundaries unaffected, in one of the constructs 17 amino acid positions altered, amounts of recombinant PPDK estimated by western blot and densitometry calculated on the basis of leaf fresh weight, wide range among individual plants Zea mays
-

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.7.9.1 additional information
-
 specific activity of PPDK in plants transformed with the construct showing 17 amino acid substitutions inversely correlates with amount of enzyme in leaves, PPDK activity becomes constant when the amount is above 5 mg/g fresh weight, enzyme contents estimated by Western blot, spectrometric assay at 25°C, two transformants retained 70% of activity after 180 min indicating a comparable cold-tolerance like Flaveria brownii plants, further effect of cold-tolerant PPDK on photosynthetic rate estimated by measurement of CO2 uptake at leaf temperatures of 30°C, 20°C, 13°C, and 8°C, increase by 23% at 8°C shown, no effects at higher temperatures Zea mays

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.9.1 ATP + pyruvate + phosphate
-
 Zea mays AMP + phosphoenolpyruvate + diphosphate
-
 r

Synonyms

EC Number Synonyms Comment Organism
2.7.9.1 PPDK
-
 Zea mays
2.7.9.1 pyruvate orthophosphate dikinase
-
 Zea mays

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
2.7.9.1 additional information
-
 cold tolerance of recombinant maize plants estimated, crude leaf extracts placed on ice to measure residual PPDK activities, PPDK extracted from untransformed maize looses 90% of its activity after 20 min, a retained PPDK activity 70% of after 180 min in two transformants corresponds to a cold-tolerance shown for Flaveria brownii plants Zea mays

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
2.7.9.1 additional information
-
 up to 70% of activity of PPDK retained after incubation of transformed leaf extracts for 180 min on ice, 5fold increased amounts of recombinant enzyme by using a construct with seventeen amino acid substitutions, PPDK extracted from untransformed maize lost 90% of its activity after 20 min, in recombinant plants the photosynthetic rate (CO2 uptake) at 8°C significantly increased by 23%, no obvious effect at higher temperatures Zea mays