Molecular and biochemical investigations of genes and enzymes involved in the phenolic metabolism of the hornwort Anthoceros agrestis
Wohl, Julia
An important group of plant compounds are phenolics, leading to many different secondary metabolites, e.g. flavonoids, lignans and lignin, cutin and suberin as well as hydroxycinnamic acid esters and amides (e.g. chlorogenic acid and rosmarinic acid). They can act for example as natural sun screens or cell wall reinforcements, essential for the conquering of dry habitats [1]. Until now, hornworts were the only gap amongst the bryophytes in understanding the phenylpropanoid metabolism on a genomic level [2, 3]. Cinnamic acid 4-hydroxylase (C4H) from Anthoceros agrestis was heterologously expressed in Escherichia coli and Physcomitrella patens. For expression in E. coli AaC4H was fused to AaCPR, a NADPH:cytochrome P450 reductase. The resulting fusion protein produced only a small amount of 4-coumaric acid. Additionally, AaCPR was present as a soluble protein and was therefore characterized after purification by metal chelate chromatography. P. patens was introduced as a suitable expression host for plant P450s. In real time PCR experiments it was shown, that expression of AaC4H was between 270 to 3700-fold compared to two potential C4Hs from P. patens. Protein extracts from transformed cultures revealed the formation of double to triple the amount of 4-coumaric acid and increased the affinity for cinnamic acid compared to the wild-type control. Two CoA-ligases, activating different (hydroxy)cinnamic and (hydroxy)benzoic acid derivatives, were found with 4-coumarate CoA-ligase (Aa4CL) and 4-hydroxybenzoate CoA-ligase (Aa4HBCL). Both differed substantially in their substrate preference. Aa4CL accepted 4-coumaric, caffeic, cinnamic, ferulic, isoferulic, 2-coumaric and 3-coumaric acid, but lacked affinity for sinapic acid and benzoic acid derivatives. The other CoA-ligase preferably activated benzoic acid or monohydroxylated benzoic acids and several other benzoic acid derivatives (except for salicylic acid, 3-aminosalicylic acid and vanillic acid) but also demonstrated activity towards cinnamic, 2-coumaric and 3-coumaric, 4-coumaric, caffeic and isoferulic acid.
Besides Aa4CL and Aa4HBCL a third potential CoA-ligase was expressed in E. coli. The amino acid sequence of Aa20832 revealed a peroxisomal signal sequence (PTS1) and two potential transmembrane helices were identified by secondary structure prediction.The protein did not activate any tested (hydroxy)cinnamic or (hydroxy)benzoic acids but demonstrated a high ATPase activity. Aa20832 might have an activity towards fatty acids, but this was not clearly proven. Another cytochrome P450 characterized in this work was AaCYP98, a hydroxycinnamoyl ester/amide 3-hydroxylase. The native AaCYP98 was expressed in P. patens and a codon optimized sequence was expressed in S. cerevisiae. AaCYP98 was able to hydroxylate 4-coumaroyl-3'-hydroxyanthranilic acid, 4-coumaroylanthranilic acid, 4-coumaroyltyramine, 4-coumaroylshikimic acid and 4-coumaroyl-4'-hydroxyphenyllactic acid but did not accept 4-coumaroylquinic acid, 4-coumaroyl-2'-threonic acid and caffeoyl-4'-hydroxyphenyllactic acid. Since these substrates were not commercially available most of them were either isolated from plant cell cultures or synthesized enzymatically or chemically. Activity of AaCYP98 was increased 13-fold after coexpression with a CPR from Coleus blumei. At last, the cytochrome P450 AaAp626 was expressed in P. patens. In a BLASTp search, the sequence demonstrated the highest identities towards putative flavonoid 3'-hydroxylases and CYP71A1. No activity was observed in enzyme assays with 4-coumaroyl-4'-hydroxyphenyllactic acid and the three flavonoids galangin, kaempferol and naringenin. Thus, the function of this protein remains unclear. In summary, based on this work the last two enzymes of the core phenylpropanoid pathway (AaC4H and Aa4CL) and enzymes of subsequent biosynthetic pathways of the hornwort Anthoceros agrestis (AaCYP98 and Aa4HBCL), as well as a NADPH-dependent CPR (AaCPR) were identified and characterized. Moreover, two proteins with yet unknown function, a P450 and another CoA-ligase, were heterologously expressed in P. patens and E. coli. These results provide a good foundation to gain more insight into the function and evolution of the plant phenylpropanoid biosynthetic pathway.
[1] Rensing (2018) Current opinion in plant biology 42: 49-54
[2] de Vries et al. (2017) Plant and Cell Physiology 58: 934-945
[3] Renault et al. (2019) Current opinion in biotechnology 56: 105-111
Philipps-Universität Marburg
Botanical sciences
https://doi.org/10.17192/z2020.0221
urn:nbn:de:hebis:04-z2020-02217
opus:9128
280
application/pdf
doctoralThesis
2020-05-28
Molecular and biochemical investigations of genes and enzymes involved in the phenolic metabolism of the hornwort Anthoceros agrestis
Hornmoos
cytochrome P450
4-coumarate CoA-ligase
4-Hydroxybenzoat CoA-Ligase
https://doi.org/10.17192/z2020.0221
urn:nbn:de:hebis:04-z2020-02217
Fachbereich Pharmazie
Pharmazeutische Biologie
2021-02-15
hornwort
2020
Zimtsäure 4-Hydroxylase
Molekulare und biochemische Untersuchung von Genen und Enzymen des Stoffwechsels phenolischer Verbindungen im Hornmoos Anthoceros agrestis
4-hydroxybenzoate CoA-ligase
An important group of plant compounds are phenolics, leading to many different secondary metabolites, e.g. flavonoids, lignans and lignin, cutin and suberin as well as hydroxycinnamic acid esters and amides (e.g. chlorogenic acid and rosmarinic acid). They can act for example as natural sun screens or cell wall reinforcements, essential for the conquering of dry habitats [1]. Until now, hornworts were the only gap amongst the bryophytes in understanding the phenylpropanoid metabolism on a genomic level [2, 3]. Cinnamic acid 4-hydroxylase (C4H) from Anthoceros agrestis was heterologously expressed in Escherichia coli and Physcomitrella patens. For expression in E. coli AaC4H was fused to AaCPR, a NADPH:cytochrome P450 reductase. The resulting fusion protein produced only a small amount of 4-coumaric acid. Additionally, AaCPR was present as a soluble protein and was therefore characterized after purification by metal chelate chromatography. P. patens was introduced as a suitable expression host for plant P450s. In real time PCR experiments it was shown, that expression of AaC4H was between 270 to 3700-fold compared to two potential C4Hs from P. patens. Protein extracts from transformed cultures revealed the formation of double to triple the amount of 4-coumaric acid and increased the affinity for cinnamic acid compared to the wild-type control. Two CoA-ligases, activating different (hydroxy)cinnamic and (hydroxy)benzoic acid derivatives, were found with 4-coumarate CoA-ligase (Aa4CL) and 4-hydroxybenzoate CoA-ligase (Aa4HBCL). Both differed substantially in their substrate preference. Aa4CL accepted 4-coumaric, caffeic, cinnamic, ferulic, isoferulic, 2-coumaric and 3-coumaric acid, but lacked affinity for sinapic acid and benzoic acid derivatives. The other CoA-ligase preferably activated benzoic acid or monohydroxylated benzoic acids and several other benzoic acid derivatives (except for salicylic acid, 3-aminosalicylic acid and vanillic acid) but also demonstrated activity towards cinnamic, 2-coumaric and 3-coumaric, 4-coumaric, caffeic and isoferulic acid.
Besides Aa4CL and Aa4HBCL a third potential CoA-ligase was expressed in E. coli. The amino acid sequence of Aa20832 revealed a peroxisomal signal sequence (PTS1) and two potential transmembrane helices were identified by secondary structure prediction.The protein did not activate any tested (hydroxy)cinnamic or (hydroxy)benzoic acids but demonstrated a high ATPase activity. Aa20832 might have an activity towards fatty acids, but this was not clearly proven. Another cytochrome P450 characterized in this work was AaCYP98, a hydroxycinnamoyl ester/amide 3-hydroxylase. The native AaCYP98 was expressed in P. patens and a codon optimized sequence was expressed in S. cerevisiae. AaCYP98 was able to hydroxylate 4-coumaroyl-3'-hydroxyanthranilic acid, 4-coumaroylanthranilic acid, 4-coumaroyltyramine, 4-coumaroylshikimic acid and 4-coumaroyl-4'-hydroxyphenyllactic acid but did not accept 4-coumaroylquinic acid, 4-coumaroyl-2'-threonic acid and caffeoyl-4'-hydroxyphenyllactic acid. Since these substrates were not commercially available most of them were either isolated from plant cell cultures or synthesized enzymatically or chemically. Activity of AaCYP98 was increased 13-fold after coexpression with a CPR from Coleus blumei. At last, the cytochrome P450 AaAp626 was expressed in P. patens. In a BLASTp search, the sequence demonstrated the highest identities towards putative flavonoid 3'-hydroxylases and CYP71A1. No activity was observed in enzyme assays with 4-coumaroyl-4'-hydroxyphenyllactic acid and the three flavonoids galangin, kaempferol and naringenin. Thus, the function of this protein remains unclear. In summary, based on this work the last two enzymes of the core phenylpropanoid pathway (AaC4H and Aa4CL) and enzymes of subsequent biosynthetic pathways of the hornwort Anthoceros agrestis (AaCYP98 and Aa4HBCL), as well as a NADPH-dependent CPR (AaCPR) were identified and characterized. Moreover, two proteins with yet unknown function, a P450 and another CoA-ligase, were heterologously expressed in P. patens and E. coli. These results provide a good foundation to gain more insight into the function and evolution of the plant phenylpropanoid biosynthetic pathway.
[1] Rensing (2018) Current opinion in plant biology 42: 49-54
[2] de Vries et al. (2017) Plant and Cell Physiology 58: 934-945
[3] Renault et al. (2019) Current opinion in biotechnology 56: 105-111
opus:9128
Philipps-Universität Marburg
cinnamic acid 4-hydroxylase
2021-02-15
ths
Prof. Dr.
Petersen
Maike
Petersen, Maike (Prof. Dr.)
Eine wichtige Gruppe pflanzlicher Inhaltstoffe sind phenolische Verbindungen, welche zu vielen unterschiedlichen Sekundärmetaboliten umgewandelt werden können, z.B. Flavonoiden, Lignanen und Lignin, Cutin und Suberin sowie Hydroxyzimtsäureestern und -amiden (z.B. Chlorogensäure und Rosmarinsäure). Diese Substanzen können zum Beispiel als natürlicher Sonnenschutz oder als Zellwandverstärkung dienen, welche für das Leben oberhalb der Wasseroberfläche unerlässlich sind [1]. Unter den Bryophyten waren die Hornmoose bisher die einzige Abteilung, bei der es keine Informationen über den Phenylpropanoid-Stoffwechsels auf genomischer Ebene gab [2, 3]. Die Zimtsäure 4-Hydroxylase aus Anthoceros agrestis wurde heterolog in Escherichia coli und Physcomitrella patens exprimiert. Zur Expression in E. coli wurde AaC4H mit AaCPR, einer NADPH:Cytochrom-P450-Reduktase, fusioniert. Das resultierende Fusionsprotein produzierte nur eine geringe Menge an 4-Cumarsäure. Zusätzlich lag AaCPR als lösliches Protein vor und konnte daher nach Aufreinigung über Affinitätschromatographie charakterisiert werden. P. patens wurde als geeigneter Expressionswirt pflanzlicher CYPs vorgestellt. In RT-qPCR Experimenten konnte gezeigt werden, dass die Expression von AaC4H zwischen 270 bis 3700-fach im Vergleich zu zwei potentiellen C4Hs aus P. patens war. Extrakte aus transformierten Kulturen zeigten die Bildung der doppelten bis dreifachen Menge an 4-Cumarsäure und erhöhten die Affinität für Zimtsäure im Vergleich zur Wildtyp-Kontrolle. Mit einer 4-Cumarat CoA-Ligase (Aa4CL) und einer 4-Hydroxybenzoat CoA-Ligase (Aa4HBCL) wurden zwei Enzyme entdeckt, welche in der Lage waren, verschiedene Zimtsäure- und Benzoesäurederivate zu aktivieren. Dabei unterschieden sich beide erheblich in ihrer Substratpräferenz. Aa4CL aktivierte ausschließlich (Hydroxy-) Zimtsäuren, akzeptierte aber keine Sinapinsäure. Aa4HBCL aktivierte vorzugsweise Benzoesäure, monohydroxylierte Benzoesäuren und viele andere Benzoesäurederivate (außer Salicylsäure, 3-Aminosalicylsäure und Vanillinsäure), zeigte aber auch Aktivität mit (Hydroxy-)Zimtsäuren (außer Ferulasäure und Sinapinsäure).
Neben Aa4CL und Aa4HBCL wurde eine dritte potenzielle CoA-Ligase in E. coli exprimiert. Die Aminosäuresequenz von Aa20832 zeigte eine peroxisomale Signalsequenz (PTS1) und zwei potenzielle Transmembran-Helices. Das Protein aktivierte weder (Hydroxy-)Zimtsäuren noch (Hydroxy-)Benzoesäuren, zeigte aber eine hohe ATPase-Aktivität. Es konnte nicht eindeutig nachgewiesen werde, ob Aa20832 eine Aktivität gegenüber Fettsäuren aufweist. Ein weiteres in dieser Arbeit charakterisiertes Enzym war AaCYP98, eine Hydroxycinnamoylester/amid 3-Hydroxylase. Eine native AaCYP98 wurde in P. patens exprimiert und eine Codon-optimierte Sequenz der AaCYP98 in S. cerevisiae. AaCYP98 war in der Lage, 4-Cumaroyl-3'-Hydroxyanthranilat, 4-Cumaroylanthranilat, 4-Cumaroyltyramin, 4-Cumaroylshikimat und 4-Cumaroyl-4'-Hydroxyphenyllactat zu hydroxylieren, nicht jedoch 4-Cumaroylchinat, 4-Cumaroyl-2'-Threonat und Caffeoyl-4'-Hydroxyphenyllactat. Da diese Substrate nicht kommerziell erhältlich waren, wurden die meisten von ihnen entweder aus pflanzlichen Zellkulturen isoliert oder enzymatisch sowie chemisch synthetisiert. Durch Koexpression einer CPR aus Coleus blumei konnte die Aktivität der AaCYP98 13-fach gesteigert werden. Das Cytochrom P450 Enzym AaAp626 wurde in P. patens exprimiert. In einer BLASTp-Suche zeigte die Sequenz die höchste Identität gegenüber putativen Flavonoid 3'-Hydroxylasen und CYP71A1. In Enzymtests mit verschiedenen Substraten konnte aber keine Aktivität beobachtet werden. Daher bleibt die Funktion dieses Proteins ungewiss. Zusammenfassend wurden in dieser Arbeit die beiden letzten Enzyme des zentralen Phenylpropanoid-Stoffwechsels (AaC4H und Aa4CL), Enzyme nachfolgender Biosynthesewege (AaCYP98 und Aa4HBCL) sowie eine NADPH-abhängige CPR (AaCPR) aus dem Hornmoos Anthoceros agrestis identifiziert und charakterisiert. Darüber hinaus wurden zwei Proteine mit noch unbekannter Funktion, ein CYP und eine weitere CoA-Ligase, heterolog in P. patens und E. coli exprimiert. Diese Ergebnisse bieten eine gute Grundlage, um einen tieferen Einblick in die Funktion und Evolution des pflanzlichen Phenylpropanoid-Biosynthesewegs zu gewinnen.
[1] Rensing (2018) Current opinion in plant biology 42: 49-54
[2] de Vries et al. (2017) Plant and Cell Physiology 58: 934-945
[3] Renault et al. (2019) Current opinion in biotechnology 56: 105-111
Phenylpropanstoffwechsel
https://archiv.ub.uni-marburg.de/diss/z2020/0221/cover.png
Kaffeesäure
4-Cumarat CoA-Ligase
monograph
English
CYP98
Publikationsserver der Universitätsbibliothek Marburg
Universitätsbibliothek Marburg
Botanical sciences
Pflanzen (Botanik)
Cytochrom P450
Wohl, Julia
Wohl
Julia
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