A structural and data-driven approach to engineering a plant cytochrome P450 enzyme
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摘要
Functional manipulation of biosynthetic enzymes such as cytochrome P450 s(or P450 s) has attracted great interest in metabolic engineering of plant natural products. Cucurbitacins and mogrosides are plant triterpenoids that share the same backbone but display contrasting bioactivities. This structural and functional diversity of the two metabolites can be manipulated by engineering P450 s. However, the functional redesign of P450 s through directed evolution(DE) or structure-guided protein engineering is time consuming and challenging, often because of a lack of high-throughput screening methods and crystal structures of P450 s. In this study, we used an integrated approach combining computational protein design, evolutionary information, and experimental data-driven optimization to alter the substrate specificity of a multifunctional P450(CYP87 D20)from cucumber. After three rounds of iterative design and evaluation of 96 protein variants, CYP87 D20, which is involved in the cucurbitacin C biosynthetic pathway, was successfully transformed into a P450 mono-oxygenase that performs a single specific hydroxylation at C11 of cucurbitadienol. This integrated P450-engineering approach can be further applied to create a de novo pathway to produce mogrol, the precursor of the natural sweetener mogroside, or to alter the structural diversity of plant triterpenoids by functionally manipulating other P450 s.
Functional manipulation of biosynthetic enzymes such as cytochrome P450 s(or P450 s) has attracted great interest in metabolic engineering of plant natural products. Cucurbitacins and mogrosides are plant triterpenoids that share the same backbone but display contrasting bioactivities. This structural and functional diversity of the two metabolites can be manipulated by engineering P450 s. However, the functional redesign of P450 s through directed evolution(DE) or structure-guided protein engineering is time consuming and challenging, often because of a lack of high-throughput screening methods and crystal structures of P450 s. In this study, we used an integrated approach combining computational protein design, evolutionary information, and experimental data-driven optimization to alter the substrate specificity of a multifunctional P450(CYP87 D20)from cucumber. After three rounds of iterative design and evaluation of 96 protein variants, CYP87 D20, which is involved in the cucurbitacin C biosynthetic pathway, was successfully transformed into a P450 mono-oxygenase that performs a single specific hydroxylation at C11 of cucurbitadienol. This integrated P450-engineering approach can be further applied to create a de novo pathway to produce mogrol, the precursor of the natural sweetener mogroside, or to alter the structural diversity of plant triterpenoids by functionally manipulating other P450 s.
Functional manipulation of biosynthetic enzymes such as cytochrome P450 s(or P450 s) has attracted great interest in metabolic engineering of plant natural products. Cucurbitacins and mogrosides are plant triterpenoids that share the same backbone but display contrasting bioactivities. This structural and functional diversity of the two metabolites can be manipulated by engineering P450 s. However, the functional redesign of P450 s through directed evolution(DE) or structure-guided protein engineering is time consuming and challenging, often because of a lack of high-throughput screening methods and crystal structures of P450 s. In this study, we used an integrated approach combining computational protein design, evolutionary information, and experimental data-driven optimization to alter the substrate specificity of a multifunctional P450(CYP87 D20)from cucumber. After three rounds of iterative design and evaluation of 96 protein variants, CYP87 D20, which is involved in the cucurbitacin C biosynthetic pathway, was successfully transformed into a P450 mono-oxygenase that performs a single specific hydroxylation at C11 of cucurbitadienol. This integrated P450-engineering approach can be further applied to create a de novo pathway to produce mogrol, the precursor of the natural sweetener mogroside, or to alter the structural diversity of plant triterpenoids by functionally manipulating other P450 s.
引文
Bornscheuer,U.T.,Huisman,G.W.,Kazlauskas,R.J.,Lutz,S.,Moore,J.C.,and Robins,K.(2012).Engineering the third wave of biocatalysis.Nature 485,185-194.
Chen,J.C.,Chiu,M.H.,Nie,R.L.,Cordell,G.A.,and Qiu,S.X.(2005).Cucurbitacins and cucurbitane glycosides:structures and biological activities.Nat Prod Rep 22,386-399.
Cherny,I.,Greisen Jr.,P.,Ashani,Y.,Khare,S.D.,Oberdorfer,G.,Leader,H.,Baker,D.,and Tawfik,D.S.(2013).Engineering V-type nerve agents detoxifying enzymes using computationally focused libraries.ACS Chem Biol 8,2394-2403.
Davis,I.W.,and Baker,D.(2009).RosettaLigand docking with full ligand and receptor flexibility.J Mol Biol 385,381-392.
Fleishman,S.J.,Leaver-Fay,A.,Corn,J.E.,Strauch,E.M.,Khare,S.D.,Koga,N.,Ashworth,J.,Murphy,P.,Richter,F.,Lemmon,G.,et al.(2011).RosettaScripts:a scripting language interface to the Rosetta macromolecular modeling suite.PLoS ONE 6,e20161.
Gerber,N.C.,and Sligar,S.G.(1992).Catalytic mechanism of cytochrome P-450:evidence for a distal charge relay.J Am Chem Soc 114,8742-8743.
Ghosh,S.(2017).Triterpene structural diversification by plant cytochrome P450 enzymes.Front Plant Sci 8,1886.
Gillam,E.M.J.,and Guengerich,F.P.(2001).Exploiting the versatility of human cytochrome P450 Enzymes:the promise of blue roses from biotechnology.IUBMB Life 52,271-277.
Goldsmith,M.,Aggarwal,N.,Ashani,Y.,Jubran,H.,Greisen,P.J.,Ovchinnikov,S.,Leader,H.,Baker,D.,Sussman,J.L.,Goldenzweig,A.,et al.(2017).Overcoming an optimization plateau in the directed evolution of highly efficient nerve agent bioscavengers.Protein Eng Des Sel 30,333-345.
Goldsmith,M.,Eckstein,S.,Ashani,Y.,Greisen,P.,Leader,H.,Sussman,J.L.,Aggarwal,N.,Ovchinnikov,S.,Tawfik,D.S.,Baker,D.,et al.(2016).Catalytic efficiencies of directly evolved phosphotriesterase variants with structurally different organophosphorus compounds in vitro.Arch Toxicol 90,2711-2724.
Govindarajan,S.,Mannervik,B.,Silverman,J.A.,Wright,K.,Regitsky,D.,Hegazy,U.,Purcell,T.J.,Welch,M.,Minshull,J.,and Gustafsson,C.(2015).Mapping of amino acid substitutions conferring herbicide resistance in wheat glutathione transferase.ACS Synth Biol 4,221-227.
Halpert,J.R.(2011).Structure and function of cytochromes P450 2B:from mechanism-based inactivators to X-ray crystal structures and back.Drug Metab Dispos 39,1113-1121.
Hanioka,N.,Gonzalez,F.J.,Lindberg,N.A.,Liu,G.,Gelboin,H.V.,and Korzekwa,K.R.(1992).Site-directed mutagenesis of cytochrome P450s CYP2A1 and CYP2A2:influence of the distal helix on the kinetics of testosterone hydroxylation.Biochemistry 31,3364-3370.
Ishigooka,M.,Shimizu,T.,Hiroya,K.,and Hatano,M.(1992).Role of Glu318 at the putative distal site in the catalytic function of cytochrome P450d.Biochemistry 31,1528-1531.
Itkin,M.,Davidovich-Rikanati,R.,Cohen,S.,Portnoy,V.,DoronFaigenboim,A.,Oren,E.,Freilich,S.,Tzuri,G.,Baranes,N.,Shen,S.,et al.(2016).The biosynthetic pathway of the nonsugar,highintensity sweetener mogroside V from Siraitia grosvenorii.Proc Natl Acad Sci USA 113,E7619-E7628.
Jung,S.T.,Lauchli,R.,and Arnold,F.H.(2011).Cytochrome P450:taming a wild type enzyme.Curr Opin Biotech 22,809-817.
Kamisetty,H.,Ovchinnikov,S.,and Baker,D.(2013).Assessing the utility of coevolution-based residue-residue contact predictions in a sequenceand structure-rich era.Proc Natl Acad Sci USA 110,15674-15679.
Kasai,R.,Nie,R.L.,Nashi,K.,Ohtani,K.,Zhou,J.,Tao,G.D.,and Tanaka,O.(1989).Sweet cucurbitane glycosides from fruits of Siraitia siamensis(chi-zi luo-han-guo),a Chinese folk medicine.Agric Biol Chem 53,3347-3349.
Khare,S.D.,Kipnis,Y.,Greisen,P.J.,Takeuchi,R.,Ashani,Y.,Goldsmith,M.,Song,Y.,Gallaher,J.L.,Silman,I.,Leader,H.,et al.(2012).Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis.Nat Chem Biol 8,294-300.
Leaver-Fay,A.,Tyka,M.,Lewis,S.M.,Lange,O.F.,Thompson,J.,Jacak,R.,et al.(2011).ROSETTA3:an object-oriented software suite for the simulation and design of macromolecules.Meth Enzymol 487,545-574.
Lemmon,G.,and Meiler,J.(2012).Rosetta Ligand docking with flexible XML protocols.In Computational Drug Discovery and Design(New York:Springer),pp.143-155.
Ma,Y.,Zhou,Y.,Ovchinnikov,S.,Greisen Jr.,P.,Huang,S.,and Shang,Y.(2016).New insights into substrate folding preference of plant OSCs.Sci Bull 61,1407-1412.
Meiler,J.,and Baker,D.(2006).ROSETTALIGAND:protein-small molecule docking with full side-chain flexibility.Proteins 65,538-548.
Monk,B.C.,Tomasiak,T.M.,Keniya,M.V.,Huschmann,F.U.,Tyndall,J.D.A.,O’Connell III,J.D.,Cannon,R.D.,McDonald,J.G.,Rodriguez,A.,Finer-Moore,J.S.,et al.(2014).Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer.Proc Natl Acad Sci USA 111,3865-3870.
Moses,T.,Pollier,J.,Thevelein,J.M.,and Goossens,A.(2013).Bioengineering of plant(tri)terpenoids:from metabolic engineering of plants to synthetic biology in vivo and in vitro.New Phytol 200,27-43.
Packer,M.S.,and Liu,D.R.(2015).Methods for the directed evolution of proteins.Nat Rev Genet 16,379-394.
Pompon,D.,Louerat,B.,Bronine,A.,and Urban,P.(1996).Yeast expression of animal and plant P450s in optimized redox environments.Meth Enzymol 272,51-64.
Reetz,M.T.(2013).Biocatalysis in organic chemistry and biotechnology:past,present,and future.J Am Chem Soc 135,12480-12496.
Remmert,M.,Biegert,A.,Hauser,A.,and S?ding,J.(2012).HHblits:lightning-fast iterative protein sequence searching by HMM-HMMalignment.Nat Methods 9,173-175.
Shang,Y.,Ma,Y.,Zhou,Y.,Zhang,H.,Duan,L.,Chen,H.,Zeng,J.,Zhou,Q.,Wang,S.,Gu,W.,et al.(2014).Biosynthesis,regulation,and domestication of bitterness in cucumber.Science 346,1084-1088.
Song,Y.,DiMaio,F.,Wang,R.Y.R.,Kim,D.,Miles,C.,Brunette,T.,Thompson,J.,and Baker,D.(2013).High-resolution comparative modeling with RosettaCM.Structure 21,1735-1742.
Swingle,W.T.(1941).Momordica grosvenori Sp.Nov.the source of the Chinese Lo Han Kuo.J Arnold Arbor 22,197-203.
Ulrich,V.,Peschke,M.,Brieke,C.,and Cryle,M.J.(2016).More than just recruitment:the X-domain influences catalysis of the first phenolic coupling reaction in A47934 biosynthesis by Cytochrome P450 StaH.Mol Biosyst 12,2992-3004.
Zerbe,K.,Pylypenko,O.,Vitali,F.,Zhang,W.,Rouset,S.,Heck,M.,Vrijbloed,J.W.,Bischoff,D.,Bister,B.,Süssmuth,R.D.,et al.(2002).Crystal structure of OxyB,a cytochrome P450 implicated in an oxidative phenol coupling reaction during vancomycin biosynthesis.JBiol Chem 277,47476-47485.
Zhang,Q.,Li,H.,Li,S.,Zhu,Y.,Zhang,G.,Zhang,H.,Zhang,W.,Shi,R.,and Zhang,C.(2012).Carboxyl formation from methyl via triple hydroxylations by XiaM in xiamycin A biosynthesis.Org Lett 14,6142-6145.
Zhou,Y.,Ma,Y.,Zeng,J.,Duan,L.,Xue,X.,Wang,H.,Lin,T.,Liu,Z.,Zeng,K.,Zhong,Y.,et al.(2016).Convergence and divergence of bitterness biosynthesis and regulation in Cucurbitaceae.Nat Plants 2,16183.
Chen,J.C.,Chiu,M.H.,Nie,R.L.,Cordell,G.A.,and Qiu,S.X.(2005).Cucurbitacins and cucurbitane glycosides:structures and biological activities.Nat Prod Rep 22,386-399.
Cherny,I.,Greisen Jr.,P.,Ashani,Y.,Khare,S.D.,Oberdorfer,G.,Leader,H.,Baker,D.,and Tawfik,D.S.(2013).Engineering V-type nerve agents detoxifying enzymes using computationally focused libraries.ACS Chem Biol 8,2394-2403.
Davis,I.W.,and Baker,D.(2009).RosettaLigand docking with full ligand and receptor flexibility.J Mol Biol 385,381-392.
Fleishman,S.J.,Leaver-Fay,A.,Corn,J.E.,Strauch,E.M.,Khare,S.D.,Koga,N.,Ashworth,J.,Murphy,P.,Richter,F.,Lemmon,G.,et al.(2011).RosettaScripts:a scripting language interface to the Rosetta macromolecular modeling suite.PLoS ONE 6,e20161.
Gerber,N.C.,and Sligar,S.G.(1992).Catalytic mechanism of cytochrome P-450:evidence for a distal charge relay.J Am Chem Soc 114,8742-8743.
Ghosh,S.(2017).Triterpene structural diversification by plant cytochrome P450 enzymes.Front Plant Sci 8,1886.
Gillam,E.M.J.,and Guengerich,F.P.(2001).Exploiting the versatility of human cytochrome P450 Enzymes:the promise of blue roses from biotechnology.IUBMB Life 52,271-277.
Goldsmith,M.,Aggarwal,N.,Ashani,Y.,Jubran,H.,Greisen,P.J.,Ovchinnikov,S.,Leader,H.,Baker,D.,Sussman,J.L.,Goldenzweig,A.,et al.(2017).Overcoming an optimization plateau in the directed evolution of highly efficient nerve agent bioscavengers.Protein Eng Des Sel 30,333-345.
Goldsmith,M.,Eckstein,S.,Ashani,Y.,Greisen,P.,Leader,H.,Sussman,J.L.,Aggarwal,N.,Ovchinnikov,S.,Tawfik,D.S.,Baker,D.,et al.(2016).Catalytic efficiencies of directly evolved phosphotriesterase variants with structurally different organophosphorus compounds in vitro.Arch Toxicol 90,2711-2724.
Govindarajan,S.,Mannervik,B.,Silverman,J.A.,Wright,K.,Regitsky,D.,Hegazy,U.,Purcell,T.J.,Welch,M.,Minshull,J.,and Gustafsson,C.(2015).Mapping of amino acid substitutions conferring herbicide resistance in wheat glutathione transferase.ACS Synth Biol 4,221-227.
Halpert,J.R.(2011).Structure and function of cytochromes P450 2B:from mechanism-based inactivators to X-ray crystal structures and back.Drug Metab Dispos 39,1113-1121.
Hanioka,N.,Gonzalez,F.J.,Lindberg,N.A.,Liu,G.,Gelboin,H.V.,and Korzekwa,K.R.(1992).Site-directed mutagenesis of cytochrome P450s CYP2A1 and CYP2A2:influence of the distal helix on the kinetics of testosterone hydroxylation.Biochemistry 31,3364-3370.
Ishigooka,M.,Shimizu,T.,Hiroya,K.,and Hatano,M.(1992).Role of Glu318 at the putative distal site in the catalytic function of cytochrome P450d.Biochemistry 31,1528-1531.
Itkin,M.,Davidovich-Rikanati,R.,Cohen,S.,Portnoy,V.,DoronFaigenboim,A.,Oren,E.,Freilich,S.,Tzuri,G.,Baranes,N.,Shen,S.,et al.(2016).The biosynthetic pathway of the nonsugar,highintensity sweetener mogroside V from Siraitia grosvenorii.Proc Natl Acad Sci USA 113,E7619-E7628.
Jung,S.T.,Lauchli,R.,and Arnold,F.H.(2011).Cytochrome P450:taming a wild type enzyme.Curr Opin Biotech 22,809-817.
Kamisetty,H.,Ovchinnikov,S.,and Baker,D.(2013).Assessing the utility of coevolution-based residue-residue contact predictions in a sequenceand structure-rich era.Proc Natl Acad Sci USA 110,15674-15679.
Kasai,R.,Nie,R.L.,Nashi,K.,Ohtani,K.,Zhou,J.,Tao,G.D.,and Tanaka,O.(1989).Sweet cucurbitane glycosides from fruits of Siraitia siamensis(chi-zi luo-han-guo),a Chinese folk medicine.Agric Biol Chem 53,3347-3349.
Khare,S.D.,Kipnis,Y.,Greisen,P.J.,Takeuchi,R.,Ashani,Y.,Goldsmith,M.,Song,Y.,Gallaher,J.L.,Silman,I.,Leader,H.,et al.(2012).Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis.Nat Chem Biol 8,294-300.
Leaver-Fay,A.,Tyka,M.,Lewis,S.M.,Lange,O.F.,Thompson,J.,Jacak,R.,et al.(2011).ROSETTA3:an object-oriented software suite for the simulation and design of macromolecules.Meth Enzymol 487,545-574.
Lemmon,G.,and Meiler,J.(2012).Rosetta Ligand docking with flexible XML protocols.In Computational Drug Discovery and Design(New York:Springer),pp.143-155.
Ma,Y.,Zhou,Y.,Ovchinnikov,S.,Greisen Jr.,P.,Huang,S.,and Shang,Y.(2016).New insights into substrate folding preference of plant OSCs.Sci Bull 61,1407-1412.
Meiler,J.,and Baker,D.(2006).ROSETTALIGAND:protein-small molecule docking with full side-chain flexibility.Proteins 65,538-548.
Monk,B.C.,Tomasiak,T.M.,Keniya,M.V.,Huschmann,F.U.,Tyndall,J.D.A.,O’Connell III,J.D.,Cannon,R.D.,McDonald,J.G.,Rodriguez,A.,Finer-Moore,J.S.,et al.(2014).Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer.Proc Natl Acad Sci USA 111,3865-3870.
Moses,T.,Pollier,J.,Thevelein,J.M.,and Goossens,A.(2013).Bioengineering of plant(tri)terpenoids:from metabolic engineering of plants to synthetic biology in vivo and in vitro.New Phytol 200,27-43.
Packer,M.S.,and Liu,D.R.(2015).Methods for the directed evolution of proteins.Nat Rev Genet 16,379-394.
Pompon,D.,Louerat,B.,Bronine,A.,and Urban,P.(1996).Yeast expression of animal and plant P450s in optimized redox environments.Meth Enzymol 272,51-64.
Reetz,M.T.(2013).Biocatalysis in organic chemistry and biotechnology:past,present,and future.J Am Chem Soc 135,12480-12496.
Remmert,M.,Biegert,A.,Hauser,A.,and S?ding,J.(2012).HHblits:lightning-fast iterative protein sequence searching by HMM-HMMalignment.Nat Methods 9,173-175.
Shang,Y.,Ma,Y.,Zhou,Y.,Zhang,H.,Duan,L.,Chen,H.,Zeng,J.,Zhou,Q.,Wang,S.,Gu,W.,et al.(2014).Biosynthesis,regulation,and domestication of bitterness in cucumber.Science 346,1084-1088.
Song,Y.,DiMaio,F.,Wang,R.Y.R.,Kim,D.,Miles,C.,Brunette,T.,Thompson,J.,and Baker,D.(2013).High-resolution comparative modeling with RosettaCM.Structure 21,1735-1742.
Swingle,W.T.(1941).Momordica grosvenori Sp.Nov.the source of the Chinese Lo Han Kuo.J Arnold Arbor 22,197-203.
Ulrich,V.,Peschke,M.,Brieke,C.,and Cryle,M.J.(2016).More than just recruitment:the X-domain influences catalysis of the first phenolic coupling reaction in A47934 biosynthesis by Cytochrome P450 StaH.Mol Biosyst 12,2992-3004.
Zerbe,K.,Pylypenko,O.,Vitali,F.,Zhang,W.,Rouset,S.,Heck,M.,Vrijbloed,J.W.,Bischoff,D.,Bister,B.,Süssmuth,R.D.,et al.(2002).Crystal structure of OxyB,a cytochrome P450 implicated in an oxidative phenol coupling reaction during vancomycin biosynthesis.JBiol Chem 277,47476-47485.
Zhang,Q.,Li,H.,Li,S.,Zhu,Y.,Zhang,G.,Zhang,H.,Zhang,W.,Shi,R.,and Zhang,C.(2012).Carboxyl formation from methyl via triple hydroxylations by XiaM in xiamycin A biosynthesis.Org Lett 14,6142-6145.
Zhou,Y.,Ma,Y.,Zeng,J.,Duan,L.,Xue,X.,Wang,H.,Lin,T.,Liu,Z.,Zeng,K.,Zhong,Y.,et al.(2016).Convergence and divergence of bitterness biosynthesis and regulation in Cucurbitaceae.Nat Plants 2,16183.