title: The substrate specificity-determining amino acid code of 4-coumarate:CoA ligase.
abstract: To reveal the structural principles determining substrate specificity of 4-coumarate:CoA ligase (4CL), the crystal structure of the phenylalanine activation domain of gramicidin S synthetase was used as a template for homology modeling. According to our model, 12 amino acid residues lining the Arabidopsis 4CL isoform 2 (At4CL2) substrate binding pocket (SBP) function as a signature motif generally determining 4CL substrate specificity. We used this substrate specificity code to create At4CL2 gain-of-function mutants. By increasing the space within the SBP we generated ferulic- and sinapic acid-activating At4CL2 variants. Increasing the hydrophobicity of the SBP resulted in At4CL2 variants with strongly enhanced conversion of cinnamic acid. These enzyme variants are suitable tools for investigating and influencing metabolic channeling mediated by 4CL. Knowledge of the 4CL specificity code will facilitate the prediction of substrate preference of numerous, still uncharacterized 4CL-like proteins.
authors.affiliation: Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, 50829 Cologne, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Schneider
authors.forename: Klaus
authors.initials: K
authors.lastname: Hövel
authors.forename: Kilian
authors.initials: K
authors.lastname: Witzel
authors.forename: Björn
authors.initials: B
authors.lastname: Hamberger
authors.forename: Dietmar
authors.initials: D
authors.lastname: Schomburg
authors.forename: Erich
authors.initials: E
authors.lastname: Kombrink
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Stuible
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002934:Cinnamates / Q000737:chemistry / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000737:chemistry* / Q000378:metabolism; D003373:Coumaric Acids / Q000378:metabolism; D015195:Drug Design; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; D002934:Cinnamates; D003373:Coumaric Acids; D020033:Protein Isoforms; C029010:cinnamic acid; C073734:sinapinic acid; C004999:ferulic acid; D003066:Coenzyme A Ligases; C019943:4-coumarate-CoA ligase