title: Structure-based engineering of benzalacetone synthase.
abstract: Benzalacetone synthase (BAS) and chalcone synthase (CHS) are plant-specific type III polyketide synthases (PKSs), sharing 70% amino acid sequence identity and highly homologous overall protein structures. BAS catalyzes the decarboxylative coupling of 4-coumaroyl-CoA with malonyl-CoA to produce the diketide benzalacetone, whereas CHS produces the tetraketide chalcone by iterative condensations with three molecules of malonyl-CoA, and folding the resulting intermediate into a new aromatic ring system. Recent crystallographic analyses of Rheum palmatum BAS revealed that the characteristic substitution of Thr132 (numbering of Medicago sativa CHS2), a conserved CHS residue lining the active-site cavity, with Leu causes steric contraction of the BAS active-site to produce the diketide, instead of the tetraketide. To test this hypothesis, we constructed a set of R. palmatum BAS site-directed mutants (L132G, L132A, L132S, L132C, L132T, L132F, L132Y, L132W and L132P), and investigated the mechanistic consequences of the point mutations. As a result, the single amino acid substitution L132T restored the chalcone-forming activity in BAS, whereas the Ala, Ser, and Cys substitutions expanded the product chain length to produce 4-coumaroyltriacetic acid lactone (CTAL) after three condensations with malonyl-CoA, but without the formation of the aromatic ring system. Homology modeling suggested that this is probably caused by the restoration of the 'coumaroyl binding pocket' in the active-site cavity. These findings provide further insights into the structural details of the catalytic mechanism of the type III PKS enzymes.
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Shimokawa
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Morita
authors.forename: Ikuro
authors.initials: I
authors.lastname: Abe
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D048630:Polyketide Synthases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D048630:Polyketide Synthases