Kinetic and physical characterisation of recombinant wild-type and mutant human protoporphyrinogen oxidases

Maneli, Mbulelo; Corrigall, Anne V.; Klump, H.; Davids, Lester M.; Kirsch, Ralph E.; Meissner, Peter N.

doi:10.1016/s1570-9639(03)00186-9

Cited by 33 publications

(58 citation statements)

References 55 publications

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“…The concentration of afliXuorfen that elicited a 50% reduction in catalytic rate was 1.48± 0.20 μM. This compares with 4.2 ±0.39μM,as reported in a previous study [11]. Using Eq.…”

Section: Resultssupporting

confidence: 74%

“…Using Eq. (3), the apparent K i was calculated as 0.53 μM acifluorfen, which compares with 0.23 μM, as determined previously [11].…”

Section: Resultsmentioning

confidence: 83%

“…The applicability for this continuous fluorescence assay for inhibition studies with human PPO was tested with acifluorfen, a competitive inhibitor with respect to the tetrapyrrole substrate [11]. Human PPO was assayed with varying concentrations of acifluorfen, and the data were fitted to Eq.…”

Section: Resultsmentioning

confidence: 99%

“…A noncovalently bound flavin adenine dinucleotide (FAD) cofactor was detected in each of these enzymes, and several were inhibited by acifluorfen, a diphenylether herbicide. Also, a recent study reported that acifluorfen is a competitive inhibitor of human PPO [11].…”

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confidence: 98%

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A continuous fluorimetric assay for protoporphyrinogen oxidase by monitoring porphyrin accumulation

Shepherd

Dailey

2005

Analytical Biochemistry

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A continuous spectro fluorimetric assay for protoporphyrinogen oxidase (PPO, EC 1.3.3.4) activity has been developed using a 96-well plate reader. Protoporphyrinogen IX, the tetrapyrrole substrate, is a colorless non fluorescent compound. The evolution of the fluorescent tetrapyrrole product, protoporphyrin IX, was detected using a fluorescence plate reader. The apparent K m (K app ) values for protoporphyrinogen IX were measured as 3.8± 0.3, 3.6± 0.5, and 1.0± 0.1 μM for the enzymes from human, Myxococcus xanthus, and Aquifex aeolicus, respectively. The K i for acifluorfen diphenylether herbicide,was measured as 0.53μM enzyme.Also, the specific activity of mouse liver mitochondrial PPO was measured as 0.043 nmol h -1 /mg mitochondria, demonstrating that this technique is useful for monitoring low-enzyme activities. This method can be used to accurately measure activities as low as 0.5 nM min -1 , representing a 50-fold increase in sensitivity over the currently used discontinuous assay. Furthermore, this continuous assay may be used to monitor up to 96 samples simultaneously. These obvious advantages over the discontinuous assay will be of importance for both the kinetic characterization of recombinant PPOs and the detection of low concentrations of this enzyme in biological samples.

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“…The concentration of afliXuorfen that elicited a 50% reduction in catalytic rate was 1.48± 0.20 μM. This compares with 4.2 ±0.39μM,as reported in a previous study [11]. Using Eq.…”

Section: Resultssupporting

confidence: 74%

“…Using Eq. (3), the apparent K i was calculated as 0.53 μM acifluorfen, which compares with 0.23 μM, as determined previously [11].…”

Section: Resultsmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 98%

See 2 more Smart Citations

A continuous fluorimetric assay for protoporphyrinogen oxidase by monitoring porphyrin accumulation

Shepherd

Dailey

2005

Analytical Biochemistry

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“…The photosensitizing porphyrin can lead to singlet oxygen, which can cause peroxidation of membrane lipids and cell death (18,19). In human, because the deficiency of PPO leads to the tissue accumulation and excessive excretion of porphyrin, it is hypothesized that the sensitivity of VP patients to light should be similar to the condition in plants (20,21).…”

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confidence: 99%

Quantitative Structural Insight into Human Variegate Porphyria Disease

Wang

Wen

Qin

et al. 2013

Journal of Biological Chemistry

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Background: Defects in the human protoporphyrinogen oxidase (PPO) causes the variegate porphyria (VP) disease. Results: The activity of 44 clinically reported VP-causing mutants have been accurately predicted. Conclusion: A quantitative understanding into the molecular basis of VP by PPO mutation was established. Significance: The quantitative insight into VP should be helpful for the diagnosis, precaution, and treatment of this disease.

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An interaction network in Bacillus subtilis coproporphyrinogen oxidase is essential for the oxidation of protoporphyrinogen IX

et al. 2023

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Coproporphyrinogen oxidase (CPO) plays important role in the biosynthesis of heme by catalyzing the coproporphyrinogen III to coproporphyrin III. However, in earlier research, it was regarded as the protoporphyrinogen oxidase (PPO) because it can also catalyze the oxidation of protoporphyrinogen IX to protoporphyrin IX. Identification of the commonalities in CPO and PPO would help us to get a further understanding of the enzyme function. In this work, we explored the role of a non‐conserved residue, Asp65 in Bacillus subtilis CPO (bsCPO), whose corresponding residues in PPO from various species are neutral or positive residue (arginine in human PPO or asparagine in tobacco PPO, etc.). We found that Asp65 performs its function by forming a polar interaction network with its surrounding residues in bsCPO, which is important for enzymatic activity. This polar network maintains the substrate binding chamber and stabilizes the micro‐environment of the isoalloxazine ring of FAD for the substrate–FAD interaction. Both the comparison of the crystal structures of bsCPO with PPO and our previous work showed that a similar polar interaction network is also present in PPOs. The results confirmed our conjecture that non‐conserved residues can form a conserved element to maintain the function of CPO or PPO.

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Kinetic and physical characterisation of recombinant wild-type and mutant human protoporphyrinogen oxidases

Cited by 33 publications

References 55 publications

A continuous fluorimetric assay for protoporphyrinogen oxidase by monitoring porphyrin accumulation

A continuous fluorimetric assay for protoporphyrinogen oxidase by monitoring porphyrin accumulation

Quantitative Structural Insight into Human Variegate Porphyria Disease

An interaction network in Bacillus subtilis coproporphyrinogen oxidase is essential for the oxidation of protoporphyrinogen IX

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