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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

Sequence and structure organization of PuuD proteins.(a) Multiple alignment of representative PuuD sequences from Agrobacterium fabrum (Af), Paracoccus denitrificans (Pd), Pseudomonas aeruginosa (Pa), Acidovorax avenae (Aa), and Ralstonia solanacearum (Rs). Accession numbers are reported in Supplementary Table S1. Colour shading of conserved residues is based on the larger alignment shown in Supplementary Fig. S7. The identified transmembrane helices in AfPuuD are indicated by gray bars and numbered with roman numerals. Blue arrowheads indicate the residues supposedly involved in heme coordination. The secondary structure elements of the cytochrome c domain are depicted above the alignment based on the comparison with the PDB structure 2d0w; helices are named according to the mitochondrial cytochrome c notation. (b) Membrane topology of AfPuuD. Residues are colored according to the Consurf analysis60 of the PuuD multiple alignment of Supplementary Fig. S7. Topology plots were obtained with the Protter program58.
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f3: Sequence and structure organization of PuuD proteins.(a) Multiple alignment of representative PuuD sequences from Agrobacterium fabrum (Af), Paracoccus denitrificans (Pd), Pseudomonas aeruginosa (Pa), Acidovorax avenae (Aa), and Ralstonia solanacearum (Rs). Accession numbers are reported in Supplementary Table S1. Colour shading of conserved residues is based on the larger alignment shown in Supplementary Fig. S7. The identified transmembrane helices in AfPuuD are indicated by gray bars and numbered with roman numerals. Blue arrowheads indicate the residues supposedly involved in heme coordination. The secondary structure elements of the cytochrome c domain are depicted above the alignment based on the comparison with the PDB structure 2d0w; helices are named according to the mitochondrial cytochrome c notation. (b) Membrane topology of AfPuuD. Residues are colored according to the Consurf analysis60 of the PuuD multiple alignment of Supplementary Fig. S7. Topology plots were obtained with the Protter program58.

Mentions: Sequences representative of the phylogenetic diversity of the PuuD family (Supplementary Fig. S6), where selected to analyse the sequence and structure conservation. The multiple alignment (Fig. 3a and Supplementary Fig. S7) shows high conservation in the N-terminal DUF989 domain, lower conservation in the Cyt_c domain and high variability in the linker region. In the N-terminal domain alignment, eight ungapped hydrophobic blocks are observed. Accordingly, the predictors of transmembrane (TM) domains identified the presence of eight strong TM helices. Consistent results were obtained using single sequences or multiple alignments (Supplementary Fig. S8a,b) and different prediction methods (Supplementary Fig. S8c). Similarly, consistent predictions were obtained for a Nout-Cout membrane topology. With this organization (Fig. 3b), the longer extra-membrane loops (specifically the loops connecting helix I to helix II and helix V to VI) are located in the cytoplasm, whereas the Cyt_c domain is located outside the plasma membrane. Cleavable signal peptides are not identified in PuuD sequences, so in these proteins helix I is assumed to function as a signal anchor for the targeting to the plasma membrane. Within the DUF989 domain, the stronger conservation is observed in helices I, II, V, VI and in the cytosolic loops I-II and V-VI. Within the Cyt_c domain, conservation pertains especially to the residues that interact with the heme cofactor. Invariant residues in all PuuD sequences are two cysteines of the CXXCH motif (Cys340 and Cys343) that covalently bind the vinyl side-chains of heme through thioether bonds plus the histidine (His344) that provides the fifth heme iron ligand. Also, invariant is a methionine residue (Met385) located about 40 residues further on towards the C-terminus, which in c-type cytochromes provides the sixth iron ligand.


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)

Sequence and structure organization of PuuD proteins.(a) Multiple alignment of representative PuuD sequences from Agrobacterium fabrum (Af), Paracoccus denitrificans (Pd), Pseudomonas aeruginosa (Pa), Acidovorax avenae (Aa), and Ralstonia solanacearum (Rs). Accession numbers are reported in Supplementary Table S1. Colour shading of conserved residues is based on the larger alignment shown in Supplementary Fig. S7. The identified transmembrane helices in AfPuuD are indicated by gray bars and numbered with roman numerals. Blue arrowheads indicate the residues supposedly involved in heme coordination. The secondary structure elements of the cytochrome c domain are depicted above the alignment based on the comparison with the PDB structure 2d0w; helices are named according to the mitochondrial cytochrome c notation. (b) Membrane topology of AfPuuD. Residues are colored according to the Consurf analysis60 of the PuuD multiple alignment of Supplementary Fig. S7. Topology plots were obtained with the Protter program58.
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Related In: Results  -  Collection

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f3: Sequence and structure organization of PuuD proteins.(a) Multiple alignment of representative PuuD sequences from Agrobacterium fabrum (Af), Paracoccus denitrificans (Pd), Pseudomonas aeruginosa (Pa), Acidovorax avenae (Aa), and Ralstonia solanacearum (Rs). Accession numbers are reported in Supplementary Table S1. Colour shading of conserved residues is based on the larger alignment shown in Supplementary Fig. S7. The identified transmembrane helices in AfPuuD are indicated by gray bars and numbered with roman numerals. Blue arrowheads indicate the residues supposedly involved in heme coordination. The secondary structure elements of the cytochrome c domain are depicted above the alignment based on the comparison with the PDB structure 2d0w; helices are named according to the mitochondrial cytochrome c notation. (b) Membrane topology of AfPuuD. Residues are colored according to the Consurf analysis60 of the PuuD multiple alignment of Supplementary Fig. S7. Topology plots were obtained with the Protter program58.
Mentions: Sequences representative of the phylogenetic diversity of the PuuD family (Supplementary Fig. S6), where selected to analyse the sequence and structure conservation. The multiple alignment (Fig. 3a and Supplementary Fig. S7) shows high conservation in the N-terminal DUF989 domain, lower conservation in the Cyt_c domain and high variability in the linker region. In the N-terminal domain alignment, eight ungapped hydrophobic blocks are observed. Accordingly, the predictors of transmembrane (TM) domains identified the presence of eight strong TM helices. Consistent results were obtained using single sequences or multiple alignments (Supplementary Fig. S8a,b) and different prediction methods (Supplementary Fig. S8c). Similarly, consistent predictions were obtained for a Nout-Cout membrane topology. With this organization (Fig. 3b), the longer extra-membrane loops (specifically the loops connecting helix I to helix II and helix V to VI) are located in the cytoplasm, whereas the Cyt_c domain is located outside the plasma membrane. Cleavable signal peptides are not identified in PuuD sequences, so in these proteins helix I is assumed to function as a signal anchor for the targeting to the plasma membrane. Within the DUF989 domain, the stronger conservation is observed in helices I, II, V, VI and in the cytosolic loops I-II and V-VI. Within the Cyt_c domain, conservation pertains especially to the residues that interact with the heme cofactor. Invariant residues in all PuuD sequences are two cysteines of the CXXCH motif (Cys340 and Cys343) that covalently bind the vinyl side-chains of heme through thioether bonds plus the histidine (His344) that provides the fifth heme iron ligand. Also, invariant is a methionine residue (Met385) located about 40 residues further on towards the C-terminus, which in c-type cytochromes provides the sixth iron ligand.

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