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Utility of a Phylogenetic Perspective in Structural Analysis of CYP72A Enzymes from Flowering Plants

View Article: PubMed Central - PubMed

ABSTRACT

Plant adaptation to external pressures depends on functional diversity in cytochrome P450 (CYP) enzymes. CYPs contain structural domains necessary for the characteristic P450 fold that allows monooxygenation, but they also have great variation in substrate binding affinity. Plant genomes typically contain hundreds of CYPs that contribute to essential functions and species-specific metabolism. The CYP72A subfamily is conserved in angiosperms but its contribution to physiological functions is largely unknown. With genomic information available for many plants, a focused analysis of CYP subfamily diversity is important to understand the contributions of these enzymes to plant evolution. This study examines the extent to which independent gene duplication and evolution have contributed to structural diversification of CYP72A enzymes in different plant lineages. CYP72A genes are prevalent across angiosperms, but the number of genes within each genome varies greatly. The prevalence of CYP72As suggest that the last common ancestor of flowering plants contained a CYP72A sequence, but gene duplication and retention has varied greatly for this CYP subfamily. Sequence comparisons show that CYP72As are involved in species-specific metabolic functions in some plants while there is likely functional conservation between closely related species. Analysis of structural and functional domains within groups of CYP72As reveals clade-specific residues that contribute to functional constraints within subsets of CYP72As. This study provides a phylogenetic framework that allows comparisons of structural features within subsets of the CYP72A subfamily. We examined a large number of sequences from a broad collection of plant species to detect patterns of functional conservation across the subfamily. The evolutionary relationships between CYPs in plant genomes are an important component in understanding the evolution of biochemical diversity in plants.

No MeSH data available.


Amino acids predicted to be in the SRS regions for CYP72A13 from Arabidopsis thaliana and CYP72A1 from Catharantheus roseus.The CYP72A13 (A) and CYP72A1 (B) substrate binding sites are highlighted with space filling spheres. All amino acids within 4 Å of the spheres are shown with some amino acids labeled for reference. (C) Amino acid sequence alignment of CYP72A13 and CYP72A1 with SRS contact residues from the models highlighted in orange and blue, respectively. Predicted SRS regions and the PERF and heme-binding domains are labeled above the sequence.
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pone.0163024.g009: Amino acids predicted to be in the SRS regions for CYP72A13 from Arabidopsis thaliana and CYP72A1 from Catharantheus roseus.The CYP72A13 (A) and CYP72A1 (B) substrate binding sites are highlighted with space filling spheres. All amino acids within 4 Å of the spheres are shown with some amino acids labeled for reference. (C) Amino acid sequence alignment of CYP72A13 and CYP72A1 with SRS contact residues from the models highlighted in orange and blue, respectively. Predicted SRS regions and the PERF and heme-binding domains are labeled above the sequence.

Mentions: In order to compare the predicted structure of the substrate binding site in different CYP72A enzymes, we modeled the structure of CYP72A1, which is a secaloganin synthase enzyme from Catharantheus roseus. The CYP72A1 structure was modeled from the crystal structure of human CYP3A4 (26% identical). The structural coordinates for the CYP72A1 model for viewing in a PDB viewer are provided in S8 Fig. This model had a template modeling score of 0.80 and an RMSD of 5.49Å, indicating a good global folding to the template [25]. As predicted for CYPs, the general fold of CYP72A1 was similar to CYP72A13, but the amino acids lining the substrate binding pocket differed more dramatically (Fig 9). Each model was searched for open pockets, and the region above the heme was highlighted with space-filling spheres (Fig 9A and 9B). The predicted binding pocket for CYP72A13 is relatively narrow over the heme and has a channel open to the outside of the enzyme through amino acids that are more N-terminal than the typical SRS regions (L25 and V22 are shown). CYP72A1 has a predicted binding pocket that is more spacious over the heme, and it has more room over the I-helix, which contains SRS4 (A367 and T371 are shown). The I-helix amino acids in CYP72A13 (F366 and A367 are shown) are oriented over the heme, thus preventing any substrate space above the I-helix. Fig 9C shows that the amino acids surrounding the modeled substrate binding pocket fall within the SRS regions predicted from previous publications and examined in Fig 7 [11,27]. The heme and PERF structural domains are not expected to contain substrate binding residues.


Utility of a Phylogenetic Perspective in Structural Analysis of CYP72A Enzymes from Flowering Plants
Amino acids predicted to be in the SRS regions for CYP72A13 from Arabidopsis thaliana and CYP72A1 from Catharantheus roseus.The CYP72A13 (A) and CYP72A1 (B) substrate binding sites are highlighted with space filling spheres. All amino acids within 4 Å of the spheres are shown with some amino acids labeled for reference. (C) Amino acid sequence alignment of CYP72A13 and CYP72A1 with SRS contact residues from the models highlighted in orange and blue, respectively. Predicted SRS regions and the PERF and heme-binding domains are labeled above the sequence.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036807&req=5

pone.0163024.g009: Amino acids predicted to be in the SRS regions for CYP72A13 from Arabidopsis thaliana and CYP72A1 from Catharantheus roseus.The CYP72A13 (A) and CYP72A1 (B) substrate binding sites are highlighted with space filling spheres. All amino acids within 4 Å of the spheres are shown with some amino acids labeled for reference. (C) Amino acid sequence alignment of CYP72A13 and CYP72A1 with SRS contact residues from the models highlighted in orange and blue, respectively. Predicted SRS regions and the PERF and heme-binding domains are labeled above the sequence.
Mentions: In order to compare the predicted structure of the substrate binding site in different CYP72A enzymes, we modeled the structure of CYP72A1, which is a secaloganin synthase enzyme from Catharantheus roseus. The CYP72A1 structure was modeled from the crystal structure of human CYP3A4 (26% identical). The structural coordinates for the CYP72A1 model for viewing in a PDB viewer are provided in S8 Fig. This model had a template modeling score of 0.80 and an RMSD of 5.49Å, indicating a good global folding to the template [25]. As predicted for CYPs, the general fold of CYP72A1 was similar to CYP72A13, but the amino acids lining the substrate binding pocket differed more dramatically (Fig 9). Each model was searched for open pockets, and the region above the heme was highlighted with space-filling spheres (Fig 9A and 9B). The predicted binding pocket for CYP72A13 is relatively narrow over the heme and has a channel open to the outside of the enzyme through amino acids that are more N-terminal than the typical SRS regions (L25 and V22 are shown). CYP72A1 has a predicted binding pocket that is more spacious over the heme, and it has more room over the I-helix, which contains SRS4 (A367 and T371 are shown). The I-helix amino acids in CYP72A13 (F366 and A367 are shown) are oriented over the heme, thus preventing any substrate space above the I-helix. Fig 9C shows that the amino acids surrounding the modeled substrate binding pocket fall within the SRS regions predicted from previous publications and examined in Fig 7 [11,27]. The heme and PERF structural domains are not expected to contain substrate binding residues.

View Article: PubMed Central - PubMed

ABSTRACT

Plant adaptation to external pressures depends on functional diversity in cytochrome P450 (CYP) enzymes. CYPs contain structural domains necessary for the characteristic P450 fold that allows monooxygenation, but they also have great variation in substrate binding affinity. Plant genomes typically contain hundreds of CYPs that contribute to essential functions and species-specific metabolism. The CYP72A subfamily is conserved in angiosperms but its contribution to physiological functions is largely unknown. With genomic information available for many plants, a focused analysis of CYP subfamily diversity is important to understand the contributions of these enzymes to plant evolution. This study examines the extent to which independent gene duplication and evolution have contributed to structural diversification of CYP72A enzymes in different plant lineages. CYP72A genes are prevalent across angiosperms, but the number of genes within each genome varies greatly. The prevalence of CYP72As suggest that the last common ancestor of flowering plants contained a CYP72A sequence, but gene duplication and retention has varied greatly for this CYP subfamily. Sequence comparisons show that CYP72As are involved in species-specific metabolic functions in some plants while there is likely functional conservation between closely related species. Analysis of structural and functional domains within groups of CYP72As reveals clade-specific residues that contribute to functional constraints within subsets of CYP72As. This study provides a phylogenetic framework that allows comparisons of structural features within subsets of the CYP72A subfamily. We examined a large number of sequences from a broad collection of plant species to detect patterns of functional conservation across the subfamily. The evolutionary relationships between CYPs in plant genomes are an important component in understanding the evolution of biochemical diversity in plants.

No MeSH data available.