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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

Experimental evidence for the urate oxidase activity of Atu2314 (COG3748).Growth curves of wild-type and engineered A. fabrum C58 strains in M9 minimal medium supplemented with (a) urate or (b) ammonia as nitrogen source. (c) Urate utilization by concentrated cell cultures. Error bars represent standard deviations obtained from three independent experiments. (d) Uox activity of 250 μg of cell-free extracts as monitored by the decrease in absorbance at 293 nm; extracts were added (arrows) to 0.1 ml solutions containing 0.11 mM urate.
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f2: Experimental evidence for the urate oxidase activity of Atu2314 (COG3748).Growth curves of wild-type and engineered A. fabrum C58 strains in M9 minimal medium supplemented with (a) urate or (b) ammonia as nitrogen source. (c) Urate utilization by concentrated cell cultures. Error bars represent standard deviations obtained from three independent experiments. (d) Uox activity of 250 μg of cell-free extracts as monitored by the decrease in absorbance at 293 nm; extracts were added (arrows) to 0.1 ml solutions containing 0.11 mM urate.

Mentions: For the anticipated difficulties in the recombinant expression and purification of the integral membrane COG3748 proteins, we sought to validate the bioinformatics prediction using a genetic approach. Agrobacterium fabrum C58 (previously known as A. tumefaciens C58) was selected as completely sequenced organism29 for which well-developed genetic tools are available3031. There is no information about urate utilization by A. fabrum, although the presence of a COG3748 gene (Atu2314, verified by sequencing of our laboratory strain), urah and urad genes (see Fig. 1c), as well as other downstream genes of the catabolic pathway32, suggest that A. fabrum is able to use urate as a nitrogen source. While in animals purine degradation is used to eliminate excess nitrogen, in plant, fungi, and prokaryotes this pathway is important for the recovery of mineral nitrogen from endogenous or imported purines3334. Accordingly, we observed growth of A. fabrum in a minimal medium supplemented with urate as the nitrogen source (Fig. 2a). With respect to the growth observed in ammonia (Fig. 2b), a longer lag was observed with urate, suggesting that in this bacterium the urate utilization system is inducible. When urate consumption was measured in highly-concentrated cell cultures, the rapid disappearance of the urate peak at 293 nm was observed after a lag of about 120 minutes (Fig. 2c). This lag was not observed with pre-induced cultures (Supplementary Fig. S4). However, no soluble urate oxidase activity could be measured using protein extracts of urate-induced cells (Fig. 2d).


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)

Experimental evidence for the urate oxidase activity of Atu2314 (COG3748).Growth curves of wild-type and engineered A. fabrum C58 strains in M9 minimal medium supplemented with (a) urate or (b) ammonia as nitrogen source. (c) Urate utilization by concentrated cell cultures. Error bars represent standard deviations obtained from three independent experiments. (d) Uox activity of 250 μg of cell-free extracts as monitored by the decrease in absorbance at 293 nm; extracts were added (arrows) to 0.1 ml solutions containing 0.11 mM urate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Experimental evidence for the urate oxidase activity of Atu2314 (COG3748).Growth curves of wild-type and engineered A. fabrum C58 strains in M9 minimal medium supplemented with (a) urate or (b) ammonia as nitrogen source. (c) Urate utilization by concentrated cell cultures. Error bars represent standard deviations obtained from three independent experiments. (d) Uox activity of 250 μg of cell-free extracts as monitored by the decrease in absorbance at 293 nm; extracts were added (arrows) to 0.1 ml solutions containing 0.11 mM urate.
Mentions: For the anticipated difficulties in the recombinant expression and purification of the integral membrane COG3748 proteins, we sought to validate the bioinformatics prediction using a genetic approach. Agrobacterium fabrum C58 (previously known as A. tumefaciens C58) was selected as completely sequenced organism29 for which well-developed genetic tools are available3031. There is no information about urate utilization by A. fabrum, although the presence of a COG3748 gene (Atu2314, verified by sequencing of our laboratory strain), urah and urad genes (see Fig. 1c), as well as other downstream genes of the catabolic pathway32, suggest that A. fabrum is able to use urate as a nitrogen source. While in animals purine degradation is used to eliminate excess nitrogen, in plant, fungi, and prokaryotes this pathway is important for the recovery of mineral nitrogen from endogenous or imported purines3334. Accordingly, we observed growth of A. fabrum in a minimal medium supplemented with urate as the nitrogen source (Fig. 2a). With respect to the growth observed in ammonia (Fig. 2b), a longer lag was observed with urate, suggesting that in this bacterium the urate utilization system is inducible. When urate consumption was measured in highly-concentrated cell cultures, the rapid disappearance of the urate peak at 293 nm was observed after a lag of about 120 minutes (Fig. 2c). This lag was not observed with pre-induced cultures (Supplementary Fig. S4). However, no soluble urate oxidase activity could be measured using protein extracts of urate-induced cells (Fig. 2d).

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