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Evolution of substrate recognition sites (SRSs) in cytochromes P450 from Apiaceae exemplified by the CYP71AJ subfamily.

Dueholm B, Krieger C, Drew D, Olry A, Kamo T, Taboureau O, Weitzel C, Bourgaud F, Hehn A, Simonsen HT - BMC Evol. Biol. (2015)

Bottom Line: Large proliferations of cytochrome P450 encoding genes resulting from gene duplications can be termed as 'blooms', providing genetic material for the genesis and evolution of biosynthetic pathways.Homology models underline a significant modification of the accession to the iron atom, which might explain the difference of the substrate specificity between the cytochromes P450 restricted to furanocoumarins as substrates and the orphan CYP71AJ.The subclades show significant variability within their substrate recognition sites between the clades, suggesting different biochemical functions and providing insights into the evolution of cytochrome P450 'blooms' in response to environmental pressures.

View Article: PubMed Central - PubMed

Affiliation: University of Copenhagen, Department of Plant and Environmental Science, Copenhagen Plant Science Centre, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark. bjorn.dueholm@mymail.unisa.edu.au.

ABSTRACT

Background: Large proliferations of cytochrome P450 encoding genes resulting from gene duplications can be termed as 'blooms', providing genetic material for the genesis and evolution of biosynthetic pathways. Furanocoumarins are allelochemicals produced by many of the species in Apiaceaous plants belonging to the Apioideae subfamily of Apiaceae and have been described as being involved in the defence reaction against phytophageous insects.

Results: A bloom in the cytochromes P450 CYP71AJ subfamily has been identified, showing at least 2 clades and 6 subclades within the CYP71AJ subfamily. Two of the subclades were functionally assigned to the biosynthesis of furanocoumarins. Six substrate recognition sites (SRS1-6) important for the enzymatic conversion were investigated in the described cytochromes P450 and display significant variability within the CYP71AJ subfamily. Homology models underline a significant modification of the accession to the iron atom, which might explain the difference of the substrate specificity between the cytochromes P450 restricted to furanocoumarins as substrates and the orphan CYP71AJ.

Conclusion: Two subclades functionally assigned to the biosynthesis of furanocoumarins and four other subclades were identified and shown to be part of two distinct clades within the CYP71AJ subfamily. The subclades show significant variability within their substrate recognition sites between the clades, suggesting different biochemical functions and providing insights into the evolution of cytochrome P450 'blooms' in response to environmental pressures.

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Potential active site configurations for five CYP71AJ groups. The homology models were constructed in CPHmodels-3.2 and VOIDOO was used for cavity calculations. SRS1: blue; SRS2 + 3: green; SRS4: yellow; SRS5: orange; SRS6: red; and heme-group: grey. The three-amino-acid motives in SRS1 that are likely important for substrate acceptance are indicated with dashed ovals. Residue differences between groups II-5 and II-6 are marked with black. Other residue likely to be important for the active sites are discussed in the text. a) CYP71AJ3, b) CYP71AJ4, c) CYP71AJ6 and CYP71AJ12, d) CYP71AJ32
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Fig4: Potential active site configurations for five CYP71AJ groups. The homology models were constructed in CPHmodels-3.2 and VOIDOO was used for cavity calculations. SRS1: blue; SRS2 + 3: green; SRS4: yellow; SRS5: orange; SRS6: red; and heme-group: grey. The three-amino-acid motives in SRS1 that are likely important for substrate acceptance are indicated with dashed ovals. Residue differences between groups II-5 and II-6 are marked with black. Other residue likely to be important for the active sites are discussed in the text. a) CYP71AJ3, b) CYP71AJ4, c) CYP71AJ6 and CYP71AJ12, d) CYP71AJ32

Mentions: Despite great variation between the enzymes in the cytochrome P450 superfamily, six substrate recognition sites (SRS1-6) have been identified constituting essential residues in the active site [18]. To investigate the potential relation between primary sequence and structure we generated homology models. The different CYP71AJ clades have distinct patterns in these six SRS regions. The SRS1 regions of the groups I-1 (VFY), I-3 (AID), II-5 (IWS), and II-6 (IWD) exhibit a “three-amino-acid” motif specific for each of these sub-clades, whereas group I-2 and I-4 share this motif (VAN) (boxed in Fig. 2, full sequence list in Additional file 2). These motif are likely be important for the substrate acceptance, and in the homology models (Fig. 4) the second residue in the motif is predicted to be in close proximity to the substrate after the final substrate orientation. The SRS1 motifs for groups II-5 and II-6 have larger residues than the residues of proteins functionally assigned to groups I-3 and I-4, which encode angelicin synthase and psoralen synthase, respectively. For all the groups, a proline localized beside amino acids harbouring large side chains (tyrosine and tryptophan) could play an important role in shaping the SRS1 region, particularly since proline residues structurally interrupt alpha helices [22]. Hydrophobic residues in the SRS1 region have been found to be conserved in cytochrome P450s described in insect herbivores feeding on furanocoumarin producing plants, e.g. in Papilio spp. feeding on apiaceous plants [23]. A similar pattern could be present in the apiaceous cytochromes P450 involved in coumarin biosynthesis.Fig. 4


Evolution of substrate recognition sites (SRSs) in cytochromes P450 from Apiaceae exemplified by the CYP71AJ subfamily.

Dueholm B, Krieger C, Drew D, Olry A, Kamo T, Taboureau O, Weitzel C, Bourgaud F, Hehn A, Simonsen HT - BMC Evol. Biol. (2015)

Potential active site configurations for five CYP71AJ groups. The homology models were constructed in CPHmodels-3.2 and VOIDOO was used for cavity calculations. SRS1: blue; SRS2 + 3: green; SRS4: yellow; SRS5: orange; SRS6: red; and heme-group: grey. The three-amino-acid motives in SRS1 that are likely important for substrate acceptance are indicated with dashed ovals. Residue differences between groups II-5 and II-6 are marked with black. Other residue likely to be important for the active sites are discussed in the text. a) CYP71AJ3, b) CYP71AJ4, c) CYP71AJ6 and CYP71AJ12, d) CYP71AJ32
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4482195&req=5

Fig4: Potential active site configurations for five CYP71AJ groups. The homology models were constructed in CPHmodels-3.2 and VOIDOO was used for cavity calculations. SRS1: blue; SRS2 + 3: green; SRS4: yellow; SRS5: orange; SRS6: red; and heme-group: grey. The three-amino-acid motives in SRS1 that are likely important for substrate acceptance are indicated with dashed ovals. Residue differences between groups II-5 and II-6 are marked with black. Other residue likely to be important for the active sites are discussed in the text. a) CYP71AJ3, b) CYP71AJ4, c) CYP71AJ6 and CYP71AJ12, d) CYP71AJ32
Mentions: Despite great variation between the enzymes in the cytochrome P450 superfamily, six substrate recognition sites (SRS1-6) have been identified constituting essential residues in the active site [18]. To investigate the potential relation between primary sequence and structure we generated homology models. The different CYP71AJ clades have distinct patterns in these six SRS regions. The SRS1 regions of the groups I-1 (VFY), I-3 (AID), II-5 (IWS), and II-6 (IWD) exhibit a “three-amino-acid” motif specific for each of these sub-clades, whereas group I-2 and I-4 share this motif (VAN) (boxed in Fig. 2, full sequence list in Additional file 2). These motif are likely be important for the substrate acceptance, and in the homology models (Fig. 4) the second residue in the motif is predicted to be in close proximity to the substrate after the final substrate orientation. The SRS1 motifs for groups II-5 and II-6 have larger residues than the residues of proteins functionally assigned to groups I-3 and I-4, which encode angelicin synthase and psoralen synthase, respectively. For all the groups, a proline localized beside amino acids harbouring large side chains (tyrosine and tryptophan) could play an important role in shaping the SRS1 region, particularly since proline residues structurally interrupt alpha helices [22]. Hydrophobic residues in the SRS1 region have been found to be conserved in cytochrome P450s described in insect herbivores feeding on furanocoumarin producing plants, e.g. in Papilio spp. feeding on apiaceous plants [23]. A similar pattern could be present in the apiaceous cytochromes P450 involved in coumarin biosynthesis.Fig. 4

Bottom Line: Large proliferations of cytochrome P450 encoding genes resulting from gene duplications can be termed as 'blooms', providing genetic material for the genesis and evolution of biosynthetic pathways.Homology models underline a significant modification of the accession to the iron atom, which might explain the difference of the substrate specificity between the cytochromes P450 restricted to furanocoumarins as substrates and the orphan CYP71AJ.The subclades show significant variability within their substrate recognition sites between the clades, suggesting different biochemical functions and providing insights into the evolution of cytochrome P450 'blooms' in response to environmental pressures.

View Article: PubMed Central - PubMed

Affiliation: University of Copenhagen, Department of Plant and Environmental Science, Copenhagen Plant Science Centre, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark. bjorn.dueholm@mymail.unisa.edu.au.

ABSTRACT

Background: Large proliferations of cytochrome P450 encoding genes resulting from gene duplications can be termed as 'blooms', providing genetic material for the genesis and evolution of biosynthetic pathways. Furanocoumarins are allelochemicals produced by many of the species in Apiaceaous plants belonging to the Apioideae subfamily of Apiaceae and have been described as being involved in the defence reaction against phytophageous insects.

Results: A bloom in the cytochromes P450 CYP71AJ subfamily has been identified, showing at least 2 clades and 6 subclades within the CYP71AJ subfamily. Two of the subclades were functionally assigned to the biosynthesis of furanocoumarins. Six substrate recognition sites (SRS1-6) important for the enzymatic conversion were investigated in the described cytochromes P450 and display significant variability within the CYP71AJ subfamily. Homology models underline a significant modification of the accession to the iron atom, which might explain the difference of the substrate specificity between the cytochromes P450 restricted to furanocoumarins as substrates and the orphan CYP71AJ.

Conclusion: Two subclades functionally assigned to the biosynthesis of furanocoumarins and four other subclades were identified and shown to be part of two distinct clades within the CYP71AJ subfamily. The subclades show significant variability within their substrate recognition sites between the clades, suggesting different biochemical functions and providing insights into the evolution of cytochrome P450 'blooms' in response to environmental pressures.

Show MeSH