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Literature summary for 1.3.3.4 extracted from

  • Boateng, M.; Corrigall, A.; Sturrock, E.; Meissner, P.
    Characterisation of the flavin adenine dinucleotide binding region of Myxococcus xanthus protoporphyrinogen oxidase (2015), Biochem. Biophys. Rep., 4, 306-311 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 1.3.3.4

Cloned(Commentary)

Cloned (Comment) Organism
gene hemeY, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain JM109, growth at 30°C (rather than at 37°C) improves expression, and yield is increased by doubling the number of cells inoculated in 1 l growth media Myxococcus xanthus

Protein Variants

Protein Variants Comment Organism
E39A site-directed mutagenesis, inactive mutant Myxococcus xanthus
E39D site-directed mutagenesis, the enzyme mutant shows 81% reduced activity compared to the wild-type enzyme Myxococcus xanthus
E39K site-directed mutagenesis, inactive mutant Myxococcus xanthus
E39Q site-directed mutagenesis, inactive mutant Myxococcus xanthus
N441I site-directed mutagenesis, the enzyme mutant shows 84% reduced activity compared to the wild-type enzyme Myxococcus xanthus
N441Q site-directed mutagenesis, the enzyme mutant shows 73% reduced activity compared to the wild-type enzyme Myxococcus xanthus
S20A site-directed mutagenesis, the enzyme mutant shows 93% reduced activity compared to the wild-type enzyme Myxococcus xanthus
S20T site-directed mutagenesis, the enzyme mutant shows 90% reduced activity compared to the wild-type enzyme Myxococcus xanthus
W408L site-directed mutagenesis, the enzyme mutant shows 48% reduced activity compared to the wild-type enzyme Myxococcus xanthus
W408Y site-directed mutagenesis, the enzyme mutant shows 82% reduced activity compared to the wild-type enzyme Myxococcus xanthus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.00038
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant wild-type enzyme Myxococcus xanthus
0.00051
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant N44I Myxococcus xanthus
0.00051
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant W408L Myxococcus xanthus
0.00059
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant E39D Myxococcus xanthus
0.00074
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant N44Q Myxococcus xanthus
0.00092
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant S20A Myxococcus xanthus
0.00117
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant S20T Myxococcus xanthus
0.00149
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant W408Y Myxococcus xanthus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
protoporphyrinogen IX + 3 O2 Myxococcus xanthus
-
 protoporphyrin IX + 3 H2O2
-
 ?

Organism

Organism UniProt Comment Textmining
Myxococcus xanthus P56601
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain JM109 by metal ion affinity chromatography Myxococcus xanthus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
protoporphyrinogen IX + 3 O2
-
 Myxococcus xanthus protoporphyrin IX + 3 H2O2
-
 ?

Synonyms

Synonyms Comment Organism
hemY
-
 Myxococcus xanthus
MxPPOX
-
 Myxococcus xanthus
PPOX
-
 Myxococcus xanthus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Myxococcus xanthus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.08
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant N44I Myxococcus xanthus
0.14
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant W408L Myxococcus xanthus
0.32
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant N44Q Myxococcus xanthus
0.32
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant W408Y Myxococcus xanthus
0.42
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant S20A Myxococcus xanthus
0.72
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant wild-type enzyme Myxococcus xanthus
0.78
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant S20T Myxococcus xanthus
1.52
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant E39D Myxococcus xanthus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8.1
-
 assay at Myxococcus xanthus

Cofactor

Cofactor Comment Organism Structure
FAD analysis of the FAD binding region and binding structures using wild-type and mutant enzymes, detailed overview. Ser20 mutants can still bind FAD, but polarity in this position is favourable, yet not essential for the integrity of FAD binding. Glu39 mutants suggest that a negative charge at position 39 is clearly favoured for interaction with the ribose ring of FAD, as all non-conservative replacements can not bind sufficient FAD. Asp441 appears not to be directly involved in FAD binding but rather in stabilizing FAD, and polarity in this position appears important. Trp408 may play a role in orientating or stabilizing the bound substrate during catalysis, and a non-polar (or slightly polar) residue is favoured at this position. Aromaticity in this position appears not to be critical Myxococcus xanthus

General Information

General Information Comment Organism
physiological function protoporphyrinogen oxidase (PPOX) i the penultimate enzyme in the heme biosynthetic pathway catalysing the six electron oxidation of protoporphyrinogen-IX to protoporphyrin-IX, in the presence of flavin adenine dinucleotide (FAD) and oxygen Myxococcus xanthus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
154.4
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant W408Y Myxococcus xanthus
156.9
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant N44I Myxococcus xanthus
274.5
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant W408L Myxococcus xanthus
426.7
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant N44Q Myxococcus xanthus
445.7
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant S20A Myxococcus xanthus
666.7
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant S20T Myxococcus xanthus
1895
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant wild-type enzyme Myxococcus xanthus
2576.3
-
 protoporphyrinogen IX pH 8.1, 37°C, recombinant mutant E39D Myxococcus xanthus