ppl12157-sup-0001-TableS1.pdfPDF document18KTable S1. List of primers used in this work. Specific use of each primer pair, name of the primer, sequence and size of the amplicon, as well as the hybridization position of each primer in the gene sequence are indicated. Underlined are shown the sequences of the restriction enzymes included in the primers for cloning into the corresponding vectors.
ppl12157-sup-0002-TableS2.pdfPDF document19KTable S2. Proteins with homology to allantoate amidohydrolases (A) and allantoicases (B). Name of the species and identification number of the protein sequence are indicated.
ppl12157-sup-0003-FigureS1.pdfPDF document69KFig. S1. Phylogenic distances among AAH (A) and allantoicases (B) Cladograms were constructed by the Neighbor-Joining method using mega5 software after multiple amino acid sequence comparison. (C) Deduced amino acid sequence of AAH from P. vulgaris (EF650088.2). The putative ER signal peptide is underlined. The putative protease and dimerisation domains are shown gray and blackshadowed, respectively. Letters in bold denote important amino acid residues, according to the enzyme from Escherichia coli.
ppl12157-sup-0004-FigureS2.pdfPDF document32KFig. S2. Alignment of the amino acid sequences of common bean, soybean and Escherichia coli AAH proteins. Deduced amino acid sequence of AAH from Phaseolus vulgaris (EF650088.2) aligned with AAH proteins from Glycine max (G. max.2, Glyma15g16870; G. max.1, Glyma09g05600), Arabidopsis thaliana (NM_116734) and E. coli (AllC). Identical and similar residues are shaded in black and gray, respectively. The putative ER signal peptide is shown underlined. Rectangles denote amino acids reported as relevant in the enzyme from E. coli for: (1) binding of metal ion; (2) binding of putative cofactor; (3) substrate binding. Putative dimerisation domain (according to E. coli AAH) is indicated by a dashed line. Protein sequences were retrieved from NCBI or Phytozome platforms and aligned using the ClustalW method.
ppl12157-sup-0005-FigureS3.pdfPDF document139KFig. S3. (A) Draft of the organization of the PvAAH genomic sequence. (B) Southern-blot for analysis of PvAAH gene copy number in the Phaseolus vulgaris genome. Introns and exons are represented by white and black boxes respectively. Copy number of PvAAH gene in the P. vulgaris genome. Southern-blot analysis of P. vulgaris genomic DNA digested with the indicated restriction enzymes and hybridized with a DIG-labeled probe of 600 bp at the 3′-end of PvAAH cDNA. Position of the probe and of the restriction sites are indicated.
ppl12157-sup-0006-FigureS4.pdfPDF document19KFig. S4. Comparison of the genomic sequence of AAH (Phvul.009g242900) and the genomic region containing the PvAAH sequenced in this work. Short 5′ and 3′ short UTR sequences are included. Amino acids encoded are placed below the nucleotide sequences of each of the exons.
ppl12157-sup-0007-FigureS5.pdfPDF document96KFig. S5. Pathway of allantoate breakdown in plants. Allantoate breakdown catalyzed by allantoate amidohydrolase yields S-ureidoglycine, which in vivo is degraded by ureidoglycine aminohydrolase, producing S-ureidoglycolate, that is acted upon by ureidoglycolate amidohydrolase, finally releasing all the nitrogen from allantoate as four ammonia molecules. S-ureidoglycine is highly unstable and might also spontaneously release glyoxylate and urea. The three enzymes catalyzing the degradation of allantoate to glyoxylate and ammonia are shown in the upper part. Products of the spontaneous decay of S-ureidoglycine are also depicted (adapted from Werner et al., 2011).

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