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The identification of an integral membrane, cytochrome c urate oxidase completes the catalytic repertoire of a therapeutic enzyme.

Doniselli N, Monzeglio E, Dal Palù A, Merli A, Percudani R - Sci Rep (2015)

Bottom Line: In contrast with the known soluble Uox, the identified gene (puuD) encodes a membrane protein with a C-terminal cytochrome c.The 8-helix transmembrane domain corresponds to DUF989, a family without similarity to known proteins.These findings identify a missing link in purine catabolism, assign a biochemical activity to a domain of unknown function (DUF989), and complete the catalytic repertoire of an enzyme useful for human therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, University of Parma, Italy.

ABSTRACT
In living organisms, the conversion of urate into allantoin requires three consecutive enzymes. The pathway was lost in hominid, predisposing humans to hyperuricemia and gout. Among other species, the genomic distribution of the two last enzymes of the pathway is wider than that of urate oxidase (Uox), suggesting the presence of unknown genes encoding Uox. Here we combine gene network analysis with association rule learning to identify the missing urate oxidase. In contrast with the known soluble Uox, the identified gene (puuD) encodes a membrane protein with a C-terminal cytochrome c. The 8-helix transmembrane domain corresponds to DUF989, a family without similarity to known proteins. Gene deletion in a PuuD-encoding organism (Agrobacterium fabrum) abolished urate degradation capacity; the phenotype was fully restored by complementation with a cytosolic Uox from zebrafish. Consistent with H2O2 production by zfUox, urate oxidation in the complemented strain caused a four-fold increase of catalase. No increase was observed in the wild-type, suggesting that urate oxidation by PuuD proceeds through cytochrome c-mediated electron transfer. These findings identify a missing link in purine catabolism, assign a biochemical activity to a domain of unknown function (DUF989), and complete the catalytic repertoire of an enzyme useful for human therapy.

No MeSH data available.


Related in: MedlinePlus

Catalase activity of PuuD and zfUox encoding strains grown on ammonia or urate.(a) Catalase activity (U/mg) of free-cell extracts obtained by sonication of urate- and ammonia-grown cells collected during the exponential growth phase; pSRKGm designates Af C58 cells transformed with the empty vector. Error bars represent standard deviation obtained from three independent experiments. (b) Urate-grown strains plated on LB agar supplemented with increasing concentrations of hydrogen peroxide. Cells were collected during exponential growth phase, diluted at OD600 = 0.1 and spotted at progressive dilutions (1, 1:5, 1:25, 1:125, 1:625). Scanned plate images were adjusted with −40% luminosity and +40% contrast.
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f4: Catalase activity of PuuD and zfUox encoding strains grown on ammonia or urate.(a) Catalase activity (U/mg) of free-cell extracts obtained by sonication of urate- and ammonia-grown cells collected during the exponential growth phase; pSRKGm designates Af C58 cells transformed with the empty vector. Error bars represent standard deviation obtained from three independent experiments. (b) Urate-grown strains plated on LB agar supplemented with increasing concentrations of hydrogen peroxide. Cells were collected during exponential growth phase, diluted at OD600 = 0.1 and spotted at progressive dilutions (1, 1:5, 1:25, 1:125, 1:625). Scanned plate images were adjusted with −40% luminosity and +40% contrast.

Mentions: As a cofactor-free oxidase, the zfUox protein used in our experiments to complement the activity of PuuD deletion mutant transfers two electrons to oxygen and generates hydrogen peroxide as a by-product of the reaction. On the other hand, the structure of the PuuD protein and the presence of an electron transfer cytochrome c domain suggest a completely different mechanism of urate oxidation. When the catalase activity was measured in protein extracts of ΔpuuD/zfUox cells, a four-fold increase of the catalase activity (p < 0.001) was observed for cells grown in urate with respect to cells grown in ammonia (Fig. 4a). Conversely, no differences in the catalase activity were observed in the parental strain in the two growth conditions. Also, no differences were observed when the parental strain was transformed with the empty vector (Fig. 4a). These results can be explained with production of H2O2 in the urate oxidation reaction catalysed by zfUox but not in the reaction catalysed by PuuD. The slight increase in the catalase activity observed in the complemented strain grown in ammonia with respect to the other strains (p < 0.1) could be explained by the degradation of endogenously produced urate. Consistent with induction of catalase by the Uox activity, we found that urate-grown ΔpuuD/zfOux strain was more resistant than the wild-type to oxidative stress, as observed by plating cells on agar medium supplemented with increasing concentrations of H2O2 (Fig. 4b).


The identification of an integral membrane, cytochrome c urate oxidase completes the catalytic repertoire of a therapeutic enzyme.

Doniselli N, Monzeglio E, Dal Palù A, Merli A, Percudani R - Sci Rep (2015)

Catalase activity of PuuD and zfUox encoding strains grown on ammonia or urate.(a) Catalase activity (U/mg) of free-cell extracts obtained by sonication of urate- and ammonia-grown cells collected during the exponential growth phase; pSRKGm designates Af C58 cells transformed with the empty vector. Error bars represent standard deviation obtained from three independent experiments. (b) Urate-grown strains plated on LB agar supplemented with increasing concentrations of hydrogen peroxide. Cells were collected during exponential growth phase, diluted at OD600 = 0.1 and spotted at progressive dilutions (1, 1:5, 1:25, 1:125, 1:625). Scanned plate images were adjusted with −40% luminosity and +40% contrast.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4562309&req=5

f4: Catalase activity of PuuD and zfUox encoding strains grown on ammonia or urate.(a) Catalase activity (U/mg) of free-cell extracts obtained by sonication of urate- and ammonia-grown cells collected during the exponential growth phase; pSRKGm designates Af C58 cells transformed with the empty vector. Error bars represent standard deviation obtained from three independent experiments. (b) Urate-grown strains plated on LB agar supplemented with increasing concentrations of hydrogen peroxide. Cells were collected during exponential growth phase, diluted at OD600 = 0.1 and spotted at progressive dilutions (1, 1:5, 1:25, 1:125, 1:625). Scanned plate images were adjusted with −40% luminosity and +40% contrast.
Mentions: As a cofactor-free oxidase, the zfUox protein used in our experiments to complement the activity of PuuD deletion mutant transfers two electrons to oxygen and generates hydrogen peroxide as a by-product of the reaction. On the other hand, the structure of the PuuD protein and the presence of an electron transfer cytochrome c domain suggest a completely different mechanism of urate oxidation. When the catalase activity was measured in protein extracts of ΔpuuD/zfUox cells, a four-fold increase of the catalase activity (p < 0.001) was observed for cells grown in urate with respect to cells grown in ammonia (Fig. 4a). Conversely, no differences in the catalase activity were observed in the parental strain in the two growth conditions. Also, no differences were observed when the parental strain was transformed with the empty vector (Fig. 4a). These results can be explained with production of H2O2 in the urate oxidation reaction catalysed by zfUox but not in the reaction catalysed by PuuD. The slight increase in the catalase activity observed in the complemented strain grown in ammonia with respect to the other strains (p < 0.1) could be explained by the degradation of endogenously produced urate. Consistent with induction of catalase by the Uox activity, we found that urate-grown ΔpuuD/zfOux strain was more resistant than the wild-type to oxidative stress, as observed by plating cells on agar medium supplemented with increasing concentrations of H2O2 (Fig. 4b).

Bottom Line: In contrast with the known soluble Uox, the identified gene (puuD) encodes a membrane protein with a C-terminal cytochrome c.The 8-helix transmembrane domain corresponds to DUF989, a family without similarity to known proteins.These findings identify a missing link in purine catabolism, assign a biochemical activity to a domain of unknown function (DUF989), and complete the catalytic repertoire of an enzyme useful for human therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, University of Parma, Italy.

ABSTRACT
In living organisms, the conversion of urate into allantoin requires three consecutive enzymes. The pathway was lost in hominid, predisposing humans to hyperuricemia and gout. Among other species, the genomic distribution of the two last enzymes of the pathway is wider than that of urate oxidase (Uox), suggesting the presence of unknown genes encoding Uox. Here we combine gene network analysis with association rule learning to identify the missing urate oxidase. In contrast with the known soluble Uox, the identified gene (puuD) encodes a membrane protein with a C-terminal cytochrome c. The 8-helix transmembrane domain corresponds to DUF989, a family without similarity to known proteins. Gene deletion in a PuuD-encoding organism (Agrobacterium fabrum) abolished urate degradation capacity; the phenotype was fully restored by complementation with a cytosolic Uox from zebrafish. Consistent with H2O2 production by zfUox, urate oxidation in the complemented strain caused a four-fold increase of catalase. No increase was observed in the wild-type, suggesting that urate oxidation by PuuD proceeds through cytochrome c-mediated electron transfer. These findings identify a missing link in purine catabolism, assign a biochemical activity to a domain of unknown function (DUF989), and complete the catalytic repertoire of an enzyme useful for human therapy.

No MeSH data available.


Related in: MedlinePlus