title: Point mutations (Q19P and N23K) increase the operational solubility of a 2alpha-O-benzoyltransferase that conveys various acyl groups from CoA to a taxane acceptor.
abstract: Two site-directed mutations within the wild-type 2-O-benzoyltransferase (tbt) cDNA, from Taxus cuspidata plants, yielded an encoded protein containing replacement amino acids at Q19P and N23K that map to a solvent-exposed loop region. The likely significant changes in the biophysical properties invoked by these mutations caused the overexpressed, modified TBT (mTBT) to partition into the soluble enzyme fraction about 5-fold greater than the wild-type enzyme. Sufficient protein could now be acquired to examine the scope of the substrate specificity of mTBT by incubation with 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III that was mixed individually with various substituted benzoyls, alkanoyls, and (E)-butenoyl CoA donors. The mTBT catalyzed the conversion of each 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III to several 7,13-O,O-diacetyl-2-O-acyl-2-O-debenzoylbaccatin III analogues. The relative catalytic efficiency of mTBT with the 7,13-O,O-diacetyl-2-O-debenzoyl surrogate substrate and heterole carbonyl CoA substrates was slightly greater than with the natural aroyl substrate benzoyl CoA, while substituted benzoyl CoA thioesters were less productive. Short-chain hydrocarbon carbonyl and cyclohexanoyl CoA thioesters were also productive, where C(4) substrates were transferred by mTBT with approximately 10- to 17-fold greater catalytic efficiency compared to the transfer of benzoyl. The described broad specificity of mTBT suggests that a plethora of 2-O-acyl variants of the antimitotic paclitaxel can be assembled through biocatalytic sequences.
authors.affiliation: Department of Chemistry and Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA.
authors.forename: Irosha N
authors.initials: IN
authors.lastname: Nawarathne
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Walker
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D001952:Bridged-Ring Compounds / Q000378:metabolism*; D015394:Molecular Structure; D017354:Point Mutation; D043823:Taxoids / Q000378:metabolism*; D020946:Taxus / Q000378:metabolism*
chemical_list: D001952:Bridged-Ring Compounds; D043823:Taxoids; C080625:taxane; C548090:3'-N-debenzoyl-2'-deoxytaxol N-benzoyltransferase, Taxus canadensis; D000217:Acyltransferases