Catalytic activity, duplication and evolution of the CYP98 cytochrome P450 family in wheat

Abstract

A burst of evolutionary duplication upon land colonization seems to have led to the large superfamily of cytochromes P450 in higher plants. Within this superfamily some clans and families are heavily duplicated. Others, such as genes involved in the phenylpropanoid pathway have led to fewer duplication events. Eight coding sequences belonging to the CYP98 family reported to catalyze the 3-hydroxylation step in this pathway were isolated from Triticum aestivum (wheat) and expressed in yeast. Comparison of the catalytic properties of the recombinant enzymes with those of CYP98s from other plant taxa was coupled to phylogenetic analyses. Our results indicate that the unusually high frequency of gene duplication in the wheat CYP98 family is a direct or indirect result from ploidization. While ancient duplication led to evolution of enzymes with different substrate preferences, most of recent duplicates underwent silencing via degenerative mutations. Three of the eight tested CYP98s from wheat have phenol meta-hydroxylase activity, with p-coumaroylshikimate being the primary substrate for all of these, as it is the case for CYP98s from sweet basil and Arabidopsis thaliana. However, CYP98s from divergent taxa have acquired different additional subsidiary activities. Some of them might be significant in the metabolism of various free or conjugated phenolics in different plant species. One of the most significant is meta-hydroxylation of p-coumaroyltyramine, predominantly by the wheat enzymes, for the synthesis of suberin phenolic monomers. Homology modeling, confirmed by directed mutagenesis, provides information on the protein regions and structural features important for some observed changes in substrate selectivity. They indicate that the metabolism of quinate ester and tyramine amide of p-coumaric acid rely on the same recognition site in the protein.

Cloning and Characterization ofLonicera japonicap-Coumaroyl Ester 3-Hydroxylase Which Is Involved in the Biosynthesis of Chlorogenic Acid

“…Most of the accessions were in two main clusters. This division into two main groups, as previously observed by Morant et al, 15) indicates that the encoding genes resulted from early duplication of an ancestral gene. LjC3H grouped in a sub-cluster of six proteins, and appeared to be closer to ABB83677 from Coffea, AAL47545 from Sesamum, ABC69348 from Nicotiana, ACC63870 from Populus, and AAS57921 from Camptotheca.…”

Isolation and mapping of a C3′H gene (CYP98A49) from globe artichoke, and its expression upon UV-C stress

“…Most of the accessions are included in two main clusters with high bootstrap probability: one comprises genes derived from Monocots species and the other one derived from Dicots and a Gymnosperm (P. taeda) species. This division into two main groups (as previously observed by Morant et al 2007) indicates that the encoding genes resulted from early duplication of ancestral gene. As opposite CYP98A8 and CYP98A9, two other CYP98A members from A. thaliana, and CYP98A20 are well separated from the main clusters.…”

“…The 3 0 -hydroxylation step is critical in the synthesis of phenolic compounds. Most of the members of the CYP98 family, described to date, metabolize shikimate esters of p-coumaric acid more efficiently than quinate esters (Schoch et al 2001(Schoch et al , 2006Morant et al 2007). On the other hand CYP98A35 from coffee is capable of metabolizing p-coumaroylquinate and p-coumaroylshikimate with the same efficiency (Mahesh et al 2007).…”

A Novel Red Clover Hydroxycinnamoyl Transferase Has Enzymatic Activities Consistent with a Role in Phaselic Acid Biosynthesis

“…We are currently characterizing the red clover C3H enzyme to determine whether it is capable of hydroxylating p-coumaroyl-malate and how likely this reaction is to occur in vivo. Even if the red clover C3H is incapable of hydroxylating p-coumaroyl-malate, it seems likely it will be capable of hydroxylating p-coumaroylshikimate like many of the already characterized C3H enzymes (Schoch et al, 2001;Franke et al, 2002;Gang et al, 2002;Morant et al, 2007). Because red clover leaves appear to have sufficiently high levels of p-coumaroylCoA:shikimate p-coumaroyl transferase activity to provide p-coumaroyl-shikimate as a hydroxylation substrate, the blue pathway in Figure 1 (top) remains plausible.…”

“…p-coumaroyl-shikimate, since several characterized C3H enzymes appear to favor this substrate [Schoch et al, 2001;Franke et al, 2002;Gang et al, 2002;Morant et al, 2007]). Following hydroxylation to the caffeoyl derivative by a C3H, the first HCT activity could synthesize caffeoyl-CoA via its reverse reaction (Hoffmann et al, 2003;Niggeweg et al, 2004).…”