Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants

Katrin Geisler; Richard K. Hughes; Frank Sainsbury; George P. Lomonossoff; Martin Rejzek; Shirley  Fairhurst; Carl Erik Olsen; Mohammed Saddik Motawie; Rachael E. Melton; Andrew M. Hemmings; Søren Bak; Anne Osbourn

doi:10.1073/pnas.1309157110

Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants

Katrin Geisler, Richard K. Hughes, Frank Sainsbury, George P. Lomonossoff, Martin Rejzek, Shirley Fairhurst, Carl Erik Olsen, Mohammed Saddik Motawie, Rachael E. Melton, Andrew M. Hemmings, Søren Bak, Anne Osbourn

Section for Plant Biochemistry

96 Citations (Scopus)

Abstract

Members of the cytochromes P450 superfamily (P450s) catalyze a huge variety of oxidation reactions in microbes and higher organisms. Most P450 families are highly divergent, but in contrast the cytochrome P450 14α-sterol demethylase (CYP51) family is one of the most ancient and conserved, catalyzing sterol 14α-demethylase reactions required for essential sterol synthesis across the fungal, animal, and plant kingdoms. Oats (Avena spp.) produce antimicrobial compounds, avenacins, that provide protection against disease. Avenacins are synthesized from the simple triterpene, β-amyrin. Previously we identified a gene encoding a member of the CYP51 family of cytochromes P450, AsCyp51H10 (also known as Saponin-deficient 2, Sad2), that is required for avenacin synthesis in a forward screen for avenacin-deficient oat mutants. sad2 mutants accumulate β-amyrin, suggesting that they are blocked early in the pathway. Here, using a transient plant expression system, we show that AsCYP51H10 is a multifunctional P450 capable of modifying both the C and D rings of the pentacyclic triterpene scaffold to give 12,13β-epoxy-3β, 16β-dihydroxy-oleanane (12,13β-epoxy-16β-hydroxy-β-amyrin). Molecular modeling and docking experiments indicate that C16 hydroxylation is likely to precede C12,13 epoxidation. Our computational modeling, in combination with analysis of a suite of sad2 mutants, provides insights into the unusual catalytic behavior of AsCYP51H10 and its active site mutants. Fungal bioassays show that the C12,13 epoxy group is an important determinant of antifungal activity. Accordingly, the oat AsCYP51H10 enzyme has been recruited from primary metabolism and has acquired a different function compared to other characterized members of the plant CYP51 family-as a multifunctional stereo- and regio-specific hydroxylase in plant specialized metabolism.

Original language	English
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	110
Issue number	35
Pages (from-to)	E3360-E3367
Number of pages	8
ISSN	0027-8424
DOIs	https://doi.org/10.1073/pnas.1309157110
Publication status	Published - 27 Aug 2013

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Geisler, K., Hughes, R. K., Sainsbury, F., Lomonossoff, G. P., Rejzek, M., Fairhurst, S., Olsen, C. E., Motawie, M. S., Melton, R. E., Hemmings, A. M., Bak, S., & Osbourn, A. (2013). Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants. Proceedings of the National Academy of Sciences of the United States of America, 110(35), E3360-E3367. https://doi.org/10.1073/pnas.1309157110

Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants. / Geisler, Katrin; Hughes, Richard K.; Sainsbury, Frank et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 110, No. 35, 27.08.2013, p. E3360-E3367.

Research output: Contribution to journal › Journal article › Research › peer-review

Geisler, K, Hughes, RK, Sainsbury, F, Lomonossoff, GP, Rejzek, M, Fairhurst, S, Olsen, CE, Motawie, MS, Melton, RE, Hemmings, AM, Bak, S & Osbourn, A 2013, 'Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants', Proceedings of the National Academy of Sciences of the United States of America, vol. 110, no. 35, pp. E3360-E3367. https://doi.org/10.1073/pnas.1309157110

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title = "Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants",

abstract = "Members of the cytochromes P450 superfamily (P450s) catalyze a huge variety of oxidation reactions in microbes and higher organisms. Most P450 families are highly divergent, but in contrast the cytochrome P450 14α-sterol demethylase (CYP51) family is one of the most ancient and conserved, catalyzing sterol 14α-demethylase reactions required for essential sterol synthesis across the fungal, animal, and plant kingdoms. Oats (Avena spp.) produce antimicrobial compounds, avenacins, that provide protection against disease. Avenacins are synthesized from the simple triterpene, β-amyrin. Previously we identified a gene encoding a member of the CYP51 family of cytochromes P450, AsCyp51H10 (also known as Saponin-deficient 2, Sad2), that is required for avenacin synthesis in a forward screen for avenacin-deficient oat mutants. sad2 mutants accumulate β-amyrin, suggesting that they are blocked early in the pathway. Here, using a transient plant expression system, we show that AsCYP51H10 is a multifunctional P450 capable of modifying both the C and D rings of the pentacyclic triterpene scaffold to give 12,13β-epoxy-3β, 16β-dihydroxy-oleanane (12,13β-epoxy-16β-hydroxy-β-amyrin). Molecular modeling and docking experiments indicate that C16 hydroxylation is likely to precede C12,13 epoxidation. Our computational modeling, in combination with analysis of a suite of sad2 mutants, provides insights into the unusual catalytic behavior of AsCYP51H10 and its active site mutants. Fungal bioassays show that the C12,13 epoxy group is an important determinant of antifungal activity. Accordingly, the oat AsCYP51H10 enzyme has been recruited from primary metabolism and has acquired a different function compared to other characterized members of the plant CYP51 family-as a multifunctional stereo- and regio-specific hydroxylase in plant specialized metabolism.",

author = "Katrin Geisler and Hughes, {Richard K.} and Frank Sainsbury and Lomonossoff, {George P.} and Martin Rejzek and Shirley Fairhurst and Olsen, {Carl Erik} and Motawie, {Mohammed Saddik} and Melton, {Rachael E.} and Hemmings, {Andrew M.} and S{\o}ren Bak and Anne Osbourn",

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T1 - Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants

AU - Geisler, Katrin

AU - Hughes, Richard K.

AU - Sainsbury, Frank

AU - Lomonossoff, George P.

AU - Rejzek, Martin

AU - Fairhurst, Shirley

AU - Olsen, Carl Erik

AU - Motawie, Mohammed Saddik

AU - Melton, Rachael E.

AU - Hemmings, Andrew M.

AU - Bak, Søren

AU - Osbourn, Anne

PY - 2013/8/27

Y1 - 2013/8/27

N2 - Members of the cytochromes P450 superfamily (P450s) catalyze a huge variety of oxidation reactions in microbes and higher organisms. Most P450 families are highly divergent, but in contrast the cytochrome P450 14α-sterol demethylase (CYP51) family is one of the most ancient and conserved, catalyzing sterol 14α-demethylase reactions required for essential sterol synthesis across the fungal, animal, and plant kingdoms. Oats (Avena spp.) produce antimicrobial compounds, avenacins, that provide protection against disease. Avenacins are synthesized from the simple triterpene, β-amyrin. Previously we identified a gene encoding a member of the CYP51 family of cytochromes P450, AsCyp51H10 (also known as Saponin-deficient 2, Sad2), that is required for avenacin synthesis in a forward screen for avenacin-deficient oat mutants. sad2 mutants accumulate β-amyrin, suggesting that they are blocked early in the pathway. Here, using a transient plant expression system, we show that AsCYP51H10 is a multifunctional P450 capable of modifying both the C and D rings of the pentacyclic triterpene scaffold to give 12,13β-epoxy-3β, 16β-dihydroxy-oleanane (12,13β-epoxy-16β-hydroxy-β-amyrin). Molecular modeling and docking experiments indicate that C16 hydroxylation is likely to precede C12,13 epoxidation. Our computational modeling, in combination with analysis of a suite of sad2 mutants, provides insights into the unusual catalytic behavior of AsCYP51H10 and its active site mutants. Fungal bioassays show that the C12,13 epoxy group is an important determinant of antifungal activity. Accordingly, the oat AsCYP51H10 enzyme has been recruited from primary metabolism and has acquired a different function compared to other characterized members of the plant CYP51 family-as a multifunctional stereo- and regio-specific hydroxylase in plant specialized metabolism.

AB - Members of the cytochromes P450 superfamily (P450s) catalyze a huge variety of oxidation reactions in microbes and higher organisms. Most P450 families are highly divergent, but in contrast the cytochrome P450 14α-sterol demethylase (CYP51) family is one of the most ancient and conserved, catalyzing sterol 14α-demethylase reactions required for essential sterol synthesis across the fungal, animal, and plant kingdoms. Oats (Avena spp.) produce antimicrobial compounds, avenacins, that provide protection against disease. Avenacins are synthesized from the simple triterpene, β-amyrin. Previously we identified a gene encoding a member of the CYP51 family of cytochromes P450, AsCyp51H10 (also known as Saponin-deficient 2, Sad2), that is required for avenacin synthesis in a forward screen for avenacin-deficient oat mutants. sad2 mutants accumulate β-amyrin, suggesting that they are blocked early in the pathway. Here, using a transient plant expression system, we show that AsCYP51H10 is a multifunctional P450 capable of modifying both the C and D rings of the pentacyclic triterpene scaffold to give 12,13β-epoxy-3β, 16β-dihydroxy-oleanane (12,13β-epoxy-16β-hydroxy-β-amyrin). Molecular modeling and docking experiments indicate that C16 hydroxylation is likely to precede C12,13 epoxidation. Our computational modeling, in combination with analysis of a suite of sad2 mutants, provides insights into the unusual catalytic behavior of AsCYP51H10 and its active site mutants. Fungal bioassays show that the C12,13 epoxy group is an important determinant of antifungal activity. Accordingly, the oat AsCYP51H10 enzyme has been recruited from primary metabolism and has acquired a different function compared to other characterized members of the plant CYP51 family-as a multifunctional stereo- and regio-specific hydroxylase in plant specialized metabolism.

U2 - 10.1073/pnas.1309157110

DO - 10.1073/pnas.1309157110

M3 - Journal article

C2 - 23940321

SN - 0027-8424

VL - 110

SP - E3360-E3367

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 35

ER -

Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants

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