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

Abstract

BackgroundLarge 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.ResultsA 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.ConclusionTwo 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0396-z) contains supplementary material, which is available to authorized users.

Semirational Protein Engineering of CYP153AM.aq.‐CPRBM3 for Efficient Terminal Hydroxylation of Short‐ to Long‐Chain Fatty Acids

“…However, a general tendency towards loss of activity was observed for most of the substitutions in the binding pocket with the three different tested fatty acids, suggesting a negative influence of mutagenesis on the substrate specificity and on the enzyme activity, although the selectivity was maintained. This has previously also been shown with six mammalian CYP subfamilies, among other studies, that reported significant impacts of the alteration of amino acids close to the prosthetic group or residues located at the entrance of the binding pocket on the substrate specificity and regioselectivity involved in substrate recognition . Our results suggest that with high selectivity towards fatty acids the binding pocket is already well optimized, and any changes probably only disturbed the favorable binding of the substrates.…”

Localization and in-Vivo Characterization of Thapsia garganica CYP76AE2 Indicates a Role in Thapsigargin Biosynthesis

“…Although the phylogeny only included P450s related to sesquiterpenoid metabolism, the sequences are from most families and subfamilies in the CYP71 clade. This shows that phylogenetic analyses are not useful to predict the specific functionality of P450s, as shown previously (Dueholm et al, 2015). The analyses merely serve to indicate the range of enzymes that have to be examined biochemically: in this case, 18 sequences.…”

“…Through the transient coexpression in Nicotiana benthamiana of TgTPS2 and TgCYP76AE2, epikunzeaol is converted to epidihydrocostunolide, a likely precursor for more complex sesquiterpenoid lactones including thapsigargin. Within the Apiaceae, only P450s involved in the biosynthesis of the phenylpropanoid coumarin have been described (Larbat et al, 2007(Larbat et al, , 2009Drew et al, 2013;Dueholm et al, 2015). This work presents the functional characterization of the first sesquiterpene-specific P450 found in the Apiaceae.…”

A bacterial artificial chromosome (BAC) genomic approach reveals partial clustering of the furanocoumarin pathway genes in parsnip

“…So far, we cannot exclude the possibility that the genes involved in the synthesis of the downstream furanocoumarin derivatives might be physically located at a different position from the first two loci identified in this work (Figure ) or even not grouped but spread all over the genome. We intend to investigate this matter further by: (i) screening other BAC clones using new probes, such as PsPT2 , another gene involved in the synthesis of osthenol (Munakata et al ., ) and also with other identified P450 genes (Dueholm et al ., ); and (ii) identifying and sequencing the clones surrounding the already identified BAC sequences.…”