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Experimental Evidence for a Revision in the Annotation of Putative Pyridoxamine 5'-Phosphate Oxidases P(N/M)P from Fungi.

Domitrovic T, Raymundo DP, da Silva TF, Palhano FL - PLoS ONE (2015)

Bottom Line: Different experimental approaches indicated that neither protein catalyzes PLP formation nor binds FMN.We suggest that a combination of two Pfam domains (PF01243 and PF10590) present in Pdx3 and other confirmed P(N/M)P oxidases would be a stronger predictor of this molecular function.This work exemplifies the importance of experimental validation to rectify genome annotation and proposes a revision in the annotation of at least 400 sequences from a wide variety of fungal species that are homologous to YLR456W and are currently misrepresented as putative P(N/M)P oxidases.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Virologia, Instituto de Microbiologia Paulo de Goes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

ABSTRACT
Pyridoxinamine 5'-phosphate oxidases (P(N/M)P oxidases) that bind flavin mononucleotide (FMN) and oxidize pyridoxine 5'-phosphate or pyridoxamine 5'-phosphate to form pyridoxal 5'-phosphate (PLP) are an important class of enzymes that play a central role in cell metabolism. Failure to generate an adequate supply of PLP is very detrimental to most organisms and is often clinically manifested as a neurological disorder in mammals. In this study, we analyzed the function of YLR456W and YPR172W, two homologous genes of unknown function from S. cerevisiae that have been annotated as putative P(N/M)P oxidases based on sequence homology. Different experimental approaches indicated that neither protein catalyzes PLP formation nor binds FMN. On the other hand, our analysis confirmed the enzymatic activity of Pdx3, the S. cerevisiae protein previously implicated in PLP biosynthesis by genetic and structural characterization. After a careful sequence analysis comparing the putative and confirmed P(N/M)P oxidases, we found that the protein domain (PF01243) that led to the YLR456W and YPR172W annotation is a poor indicator of P(N/M)P oxidase activity. We suggest that a combination of two Pfam domains (PF01243 and PF10590) present in Pdx3 and other confirmed P(N/M)P oxidases would be a stronger predictor of this molecular function. This work exemplifies the importance of experimental validation to rectify genome annotation and proposes a revision in the annotation of at least 400 sequences from a wide variety of fungal species that are homologous to YLR456W and are currently misrepresented as putative P(N/M)P oxidases.

No MeSH data available.


Related in: MedlinePlus

Ylr456w and Ypr172w annotations are inferred from only one domain involved in pyridoxamine 5’-phosphate function.(A) Pfam protein family database profile of the proteins Ylr456w, Ypr172w and Pdx3. PF01243 Pyridox_oxidase domain and PF10590 Pyridox_oxidase C-terminal dimerization domain are located on the N-terminal and C-terminal, respectively. (B) Vitamin B6 biosynthesis. (C) Crystal structure of the P(N/M)P oxidase Pdx3 (pdb 1CI0) from S. cerevisiae. The P(N/M)P oxidase protein is a homodimer (monomers colored in cyan and magenta) that binds two FMN molecules. The side chain of the conserved residues that contact FMN and their ligands are shown in black (residues R73, L76, K96, Q153, S154, E197, W199, R205, H207, R209 and P228 are indicated by red asterisks in Fig 4). The second structure (bottom) was colored to show the tridimensional position of the amino-acid residues within PF01243 in green and PF10590 in red.
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pone.0136761.g001: Ylr456w and Ypr172w annotations are inferred from only one domain involved in pyridoxamine 5’-phosphate function.(A) Pfam protein family database profile of the proteins Ylr456w, Ypr172w and Pdx3. PF01243 Pyridox_oxidase domain and PF10590 Pyridox_oxidase C-terminal dimerization domain are located on the N-terminal and C-terminal, respectively. (B) Vitamin B6 biosynthesis. (C) Crystal structure of the P(N/M)P oxidase Pdx3 (pdb 1CI0) from S. cerevisiae. The P(N/M)P oxidase protein is a homodimer (monomers colored in cyan and magenta) that binds two FMN molecules. The side chain of the conserved residues that contact FMN and their ligands are shown in black (residues R73, L76, K96, Q153, S154, E197, W199, R205, H207, R209 and P228 are indicated by red asterisks in Fig 4). The second structure (bottom) was colored to show the tridimensional position of the amino-acid residues within PF01243 in green and PF10590 in red.

Mentions: In this study, we used different experimental approaches to verify the annotation of two unknown yeast genes, YLR456W and its homolog YPR172W. These two genes are closely related and were originated by genome duplication [10]. Both are computationally assigned to the GO term “pyridoxamine phosphate oxidase” in SGD (Fig 1A, Pfam01243). This class of enzymes is involved in vitamin B6 metabolism and plays a central role in amino-acid biosynthesis and other important pathways [11]. Vitamin B6 is a generic term for pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM) and their related phosphorylated forms (PNP, PLP and PMP, respectively). Pyridoxamine phosphate oxidases are enzymes that convert PMP or PNP into PLP (Fig 1B), and most PNP oxidases possess PMP oxidase activity as well, so they are known as P(N/M)P oxidases (Fig 1B). PLP is the most versatile organic cofactor in biology, and production deficiencies in mammals are often clinically manifested as neurological disorders [11]. All P(N/M)P oxidases form stable dimers that contain an FMN-binding split barrel fold and require flavin mononucleotide (FMN) to be active [12].


Experimental Evidence for a Revision in the Annotation of Putative Pyridoxamine 5'-Phosphate Oxidases P(N/M)P from Fungi.

Domitrovic T, Raymundo DP, da Silva TF, Palhano FL - PLoS ONE (2015)

Ylr456w and Ypr172w annotations are inferred from only one domain involved in pyridoxamine 5’-phosphate function.(A) Pfam protein family database profile of the proteins Ylr456w, Ypr172w and Pdx3. PF01243 Pyridox_oxidase domain and PF10590 Pyridox_oxidase C-terminal dimerization domain are located on the N-terminal and C-terminal, respectively. (B) Vitamin B6 biosynthesis. (C) Crystal structure of the P(N/M)P oxidase Pdx3 (pdb 1CI0) from S. cerevisiae. The P(N/M)P oxidase protein is a homodimer (monomers colored in cyan and magenta) that binds two FMN molecules. The side chain of the conserved residues that contact FMN and their ligands are shown in black (residues R73, L76, K96, Q153, S154, E197, W199, R205, H207, R209 and P228 are indicated by red asterisks in Fig 4). The second structure (bottom) was colored to show the tridimensional position of the amino-acid residues within PF01243 in green and PF10590 in red.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556617&req=5

pone.0136761.g001: Ylr456w and Ypr172w annotations are inferred from only one domain involved in pyridoxamine 5’-phosphate function.(A) Pfam protein family database profile of the proteins Ylr456w, Ypr172w and Pdx3. PF01243 Pyridox_oxidase domain and PF10590 Pyridox_oxidase C-terminal dimerization domain are located on the N-terminal and C-terminal, respectively. (B) Vitamin B6 biosynthesis. (C) Crystal structure of the P(N/M)P oxidase Pdx3 (pdb 1CI0) from S. cerevisiae. The P(N/M)P oxidase protein is a homodimer (monomers colored in cyan and magenta) that binds two FMN molecules. The side chain of the conserved residues that contact FMN and their ligands are shown in black (residues R73, L76, K96, Q153, S154, E197, W199, R205, H207, R209 and P228 are indicated by red asterisks in Fig 4). The second structure (bottom) was colored to show the tridimensional position of the amino-acid residues within PF01243 in green and PF10590 in red.
Mentions: In this study, we used different experimental approaches to verify the annotation of two unknown yeast genes, YLR456W and its homolog YPR172W. These two genes are closely related and were originated by genome duplication [10]. Both are computationally assigned to the GO term “pyridoxamine phosphate oxidase” in SGD (Fig 1A, Pfam01243). This class of enzymes is involved in vitamin B6 metabolism and plays a central role in amino-acid biosynthesis and other important pathways [11]. Vitamin B6 is a generic term for pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM) and their related phosphorylated forms (PNP, PLP and PMP, respectively). Pyridoxamine phosphate oxidases are enzymes that convert PMP or PNP into PLP (Fig 1B), and most PNP oxidases possess PMP oxidase activity as well, so they are known as P(N/M)P oxidases (Fig 1B). PLP is the most versatile organic cofactor in biology, and production deficiencies in mammals are often clinically manifested as neurological disorders [11]. All P(N/M)P oxidases form stable dimers that contain an FMN-binding split barrel fold and require flavin mononucleotide (FMN) to be active [12].

Bottom Line: Different experimental approaches indicated that neither protein catalyzes PLP formation nor binds FMN.We suggest that a combination of two Pfam domains (PF01243 and PF10590) present in Pdx3 and other confirmed P(N/M)P oxidases would be a stronger predictor of this molecular function.This work exemplifies the importance of experimental validation to rectify genome annotation and proposes a revision in the annotation of at least 400 sequences from a wide variety of fungal species that are homologous to YLR456W and are currently misrepresented as putative P(N/M)P oxidases.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Virologia, Instituto de Microbiologia Paulo de Goes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

ABSTRACT
Pyridoxinamine 5'-phosphate oxidases (P(N/M)P oxidases) that bind flavin mononucleotide (FMN) and oxidize pyridoxine 5'-phosphate or pyridoxamine 5'-phosphate to form pyridoxal 5'-phosphate (PLP) are an important class of enzymes that play a central role in cell metabolism. Failure to generate an adequate supply of PLP is very detrimental to most organisms and is often clinically manifested as a neurological disorder in mammals. In this study, we analyzed the function of YLR456W and YPR172W, two homologous genes of unknown function from S. cerevisiae that have been annotated as putative P(N/M)P oxidases based on sequence homology. Different experimental approaches indicated that neither protein catalyzes PLP formation nor binds FMN. On the other hand, our analysis confirmed the enzymatic activity of Pdx3, the S. cerevisiae protein previously implicated in PLP biosynthesis by genetic and structural characterization. After a careful sequence analysis comparing the putative and confirmed P(N/M)P oxidases, we found that the protein domain (PF01243) that led to the YLR456W and YPR172W annotation is a poor indicator of P(N/M)P oxidase activity. We suggest that a combination of two Pfam domains (PF01243 and PF10590) present in Pdx3 and other confirmed P(N/M)P oxidases would be a stronger predictor of this molecular function. This work exemplifies the importance of experimental validation to rectify genome annotation and proposes a revision in the annotation of at least 400 sequences from a wide variety of fungal species that are homologous to YLR456W and are currently misrepresented as putative P(N/M)P oxidases.

No MeSH data available.


Related in: MedlinePlus