RESUMO
A cDNA encoding a protein with 456 amino acids whose sequence shows considerable similarity to plant acyltransferases was identified among 750 Clarkia breweri flower expressed sequence tags. The cDNA was expressed in Escherichia coli, and the protein produced was shown to encode the enzyme benzoyl-coenzyme A (CoA):benzyl alcohol benzoyl transferase (BEBT). BEBT catalyzes the formation of benzylbenzoate, a minor constituent of the C. breweri floral aroma, but it also has activity with a number of other alcohols and acyl CoAs. The BEBT gene is expressed in different parts of the flowers with maximal RNA transcript levels in the stigma, and no expression was observed in the leaves under normal conditions. However, BEBT expression was induced in damaged leaves, reaching a maximum 6 h after damage occurred. We also show here that a closely related tobacco (Nicotiana tabacum) gene previously shown to be induced in leaves after being challenged by phytopathogenic bacteria also has BEBT activity, whereas the most similar protein to BEBT in the Arabidopsis proteome does not use benzoyl CoA as a substrate and instead can use acetyl CoA to catalyze the formation of cis-3-hexen-1-yl acetate, a green-leaf volatile.
Assuntos
Aciltransferases/genética , Benzoatos/metabolismo , Onagraceae/enzimologia , Aciltransferases/isolamento & purificação , Aciltransferases/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , DNA Complementar/química , DNA Complementar/genética , Escherichia coli/genética , Ésteres , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Immunoblotting , Cinética , Dados de Sequência Molecular , Onagraceae/genética , Onagraceae/metabolismo , Filogenia , Folhas de Planta/metabolismo , Caules de Planta/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Estresse Mecânico , Especificidade por Substrato , Nicotiana/genética , VolatilizaçãoRESUMO
Benzoate:CoA ligase (BZL) was partially purified from flowers of the annual California plant Clarkia breweri. BZL catalyzes the formation of benzoyl-CoA and anthraniloyl-CoA, important intermediates for subsequent acyltransferase reactions in plant secondary metabolism. The native enzyme is active as a monomer with a molecular mass of approximately 59-64.5 kDa, and it has K(m) values of 45, 95, and 130 microM for benzoic acid, ATP, and CoA, respectively. BZL is most active in the pH range of 7.2-8.4, and its activity is strictly dependent on certain bivalent cations. BZL is an AMP-forming enzyme. Overall, its properties suggest that it is related to the family of CoA ligase enzymes that includes the plant enzyme 4-hydroxycinnamate:CoA ligase.
