title: Paclitaxel Biosynthesis: Adenylation and Thiolation Domains of an NRPS TycA PheAT Module Produce Various Arylisoserine CoA Thioesters.
abstract: Structure-activity relationship studies show that the phenylisoserinyl moiety of paclitaxel (Taxol) is largely necessary for the effective anticancer activity. Several paclitaxel analogues with a variant isoserinyl side chain have improved pharmaceutical properties versus those of the parent drug. To produce the isoserinyl CoAs as intermediates needed for enzyme catalysis on a semibiosynthetic pathway to paclitaxel analogues, we repurposed the adenylation and thiolation domains (Phe-AT) of a nonribosomal peptide synthetase (TycA) so that they would function as a CoA ligase. Twenty-eight isoserine analogue racemates were synthesized by an established procedure based on the Staudinger [2+2] cycloaddition reaction. Phe-AT converted 16 substituted phenylisoserines, one β-(heteroaryl)isoserine, and one β-(cyclohexyl)isoserine to their corresponding isoserinyl CoAs. We imagine that these CoA thioesters can likely serve as linchpin biosynthetic acyl donors transferred by a 13-O-acyltransferase to a paclitaxel precursor baccatin III to make drug analogues with better efficacy. It was also interesting to find that an active site mutant [Phe-AT (W227S)] turned over 2-pyridylisoserine and the sterically demanding p-methoxyphenylisoserine substrates to their CoA thioesters, while Phe-AT did not. This mutant is promising for further development to make 3-fluoro-2-pyridylisoserinyl CoA, a biosynthetic precursor of the oral pharmaceutical tesetaxel used for gastric cancers.
authors.affiliation: Department of Chemistry, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Ruth
authors.initials: R
authors.lastname: Muchiri
authors.affiliation: Department of Chemistry, Michigan State University , East Lansing, Michigan 48824, United States.|Department of Biochemistry and Molecular Biology, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Kevin D
authors.identifier: 0000-0001-5208-6692
authors.initials: KD
authors.lastname: Walker
mesh_terms: D000470:Alkaloids / Q000096:biosynthesis / Q000138:chemical synthesis; D000972:Antineoplastic Agents, Phytogenic / Q000096:biosynthesis* / Q000138:chemical synthesis; D058675:Brevibacillus / Q000737:chemistry / Q000201:enzymology; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003065:Coenzyme A / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D017239:Paclitaxel / Q000096:biosynthesis / Q000138:chemical synthesis; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D000072417:Protein Domains; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D043823:Taxoids / Q000138:chemical synthesis / Q000378:metabolism; D020946:Taxus / Q000737:chemistry / Q000201:enzymology
chemical_list: D000470:Alkaloids; D000972:Antineoplastic Agents, Phytogenic; D010940:Plant Proteins; D011994:Recombinant Proteins; D043823:Taxoids; C586689:phenylisoserinyl CoA, Taxus; C073950:baccatin III; D010453:Peptide Synthases; C035180:tyrocidine synthetase; D017239:Paclitaxel; D003065:Coenzyme A; C479543:tesetaxel