OH R O NH 2 PheAT SCoA R O NH 2 OH OH ATP CoASH AMP PP i X = CH, Y = F, CH 3 , Cl, Br, OH, OCH 3 , NO 2 Bold = Productive Substrate X = N and Y = F (pending) Z o = F, CH 3 , Cl, NO 2 Z m = F, CH 3 , Cl, Br, OH, OCH 3 NO 2 R= X Y Z m Z o Semi-Biosynthesis of Taxol and Its Analogs from Baccatin III Precursors in Four Enzymatic Steps 1. Phenylisoserine CoA Ligase (Repurposing an NRPS) 2. O-Phenylpropanoyl Transferase (BAPT) 3. Benzoate CoA Ligase (BadA) 4. N-Benzoyltransferase (NDTBT) PheAT (phenylpropanoate:CoA ligase): makes isoserinyl CoAs BAPT (A Taxus β-phenylpropanoyl transferase) conveys phenylisoserinyl to baccatin III A benzoate:CoA ligase (BadA) from Rhodopseudomonas palustris 6 makes several acyl CoA thioesters for biosynthesis of Taxol analogues. BadA structure 1.8 Å (pdb: 4EAT) PheAT Homology Model (Swiss-Model) NDTBT (A Taxus N-debenzoylpaclitaxel N- benzoyltransferase) catalyzes the final step and is permissive with several different acyl CoA substrates. NDTBT Homology Model (Raptor X) BAPT Homology Model (Raptor X) R O OH ATP, Mg 2+ CoASH AMP R O SCoA PP i BadA X X X X = H, F, OH, NH 2 , CH 3 , OCH 3 , CN, ortho X = NO 2 Z Y X = S, Y = C X = O, Y = C X = C, Y = O R= O O O O OH O H O OH 2' 3' OH NH O O O O O UNDERGRADUATES CAN EMBARK ON STUDIES INVOLVING ORGANIC SYNTHESIS OF NOVEL ENZYME SUBSTRATES, MOLECULAR CLONING TECHNIQUES, EXPRESSION OF VARIOUS ENZYMES IN BACTERIA, ASSAY DEVELOPMENT, BASIC BIOCHEMISTRY AND MOLECULAR BIOLOGICAL APPROACHES RELATED TO ENZYME KINETICS, ENZYME PURIFICATION AND CHARACTERIZATION.OTHER AREAS INVOLVE VARIOUS ANALYTICAL TECHNIQUES (NMR, GC/MS, LC-MS(/MS), ETC.). Undergraduate Research Opportunity in the Walker Lab Walker Lab Research Overview Department of Chemistry and Department of Biochemistry and Molecular Biology Michigan State University, East Lansing, MI 48842, United States

Semi-Biosynthesis of Taxol and Its Analogs ... - REU Program · ROH NH 2 O PheAT SCoA NH OH ATP CoASH AMP PP i X=CH,Y=F, CH 3,Cl,Br,OH,OCH3, NO 2 Bold = Productive Substrate X = N

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Page 1: Semi-Biosynthesis of Taxol and Its Analogs ... - REU Program · ROH NH 2 O PheAT SCoA NH OH ATP CoASH AMP PP i X=CH,Y=F, CH 3,Cl,Br,OH,OCH3, NO 2 Bold = Productive Substrate X = N

OHR

ONH2

PheAT

SCoAR

ONH2

OHOH

ATPCoASH

AMPPPi

X = CH, Y = F, CH3, Cl, Br, OH, OCH3, NO2

Bold = Productive Substrate

X = N and Y = F (pending)Zo = F, CH3, Cl, NO2Zm = F, CH3, Cl, Br, OH, OCH3 NO2

R = X

Semi-Biosynthesis of Taxol and Its Analogs from Baccatin III Precursors in Four Enzymatic Steps

1. Phenylisoserine CoA Ligase (Repurposing an NRPS) 2. O-Phenylpropanoyl Transferase (BAPT)

3. Benzoate CoA Ligase (BadA) 4. N-Benzoyltransferase (NDTBT)

PheAT (phenylpropanoate:CoA ligase): makes isoserinyl CoAs BAPT (A Taxus β-phenylpropanoyl transferase) conveysphenylisoserinyl to baccatin III

A benzoate:CoA ligase (BadA) from Rhodopseudomonaspalustris6 makes several acyl CoA thioesters forbiosynthesis of Taxol analogues.

BadA structure1.8 Å (pdb: 4EAT)

PheAT Homology Model (Swiss-Model)

NDTBT (A Taxus N-debenzoylpaclitaxel N-benzoyltransferase) catalyzes the final step and is permissivewith several different acyl CoA substrates.

NDTBT Homology Model(Raptor X)

BAPT Homology Model(Raptor X)

ATP, Mg2+CoASH

AMPR

SCoA

PPi

BadA

X = H, F, OH, NH2, CH3, OCH3,CN, ortho X = NO2

X = S, Y = CX = O, Y = CX = C, Y = O

R =

OOH

2'3'

NH O

UNDERGRADUATES CAN EMBARK ON STUDIES INVOLVING ORGANIC SYNTHESIS OF NOVEL ENZYME SUBSTRATES, MOLECULAR CLONINGTECHNIQUES, EXPRESSION OF VARIOUS ENZYMES IN BACTERIA, ASSAY DEVELOPMENT, BASIC BIOCHEMISTRY AND MOLECULAR BIOLOGICALAPPROACHES RELATED TO ENZYME KINETICS, ENZYME PURIFICATION AND CHARACTERIZATION. OTHER AREAS INVOLVE VARIOUS ANALYTICALTECHNIQUES (NMR, GC/MS, LC-MS(/MS), ETC.).

Undergraduate Research Opportunity in the Walker Lab

Walker Lab Research OverviewDepartment of Chemistry and Department of Biochemistry and Molecular Biology

Michigan State University, East Lansing, MI 48842, United States