RESUMO
Primary long-chain fatty alcohols are present in a variety of phyla. In eukaryotes, the production of fatty alcohols is catalyzed by fatty acyl-CoA reductase (FAR) enzymes that convert fatty acyl-CoAs or acyl-ACPs into fatty alcohols. Here, we report on the biochemical properties of a purified plant FAR, Arabidopsis FAR6 (AtFAR6). In vitro assays show that the enzyme preferentially uses 16 carbon acyl-chains as substrates and produces predominantly fatty alcohols. Free fatty acids and fatty aldehyde intermediates can be released from the enzyme, in particular with suboptimal chain lengths and concentrations of the substrates. Both acyl-CoA and acyl-ACP could serve as substrates. Transient expression experiments in Nicotiana tabacum showed that AtFAR6 is a chloroplast localized FAR. In addition, expression of full length AtFAR6 in Nicotiana benthamiana leaves resulted in the production of C16:0-alcohol within this organelle. Finally, a GUS reporter gene fusion with the AtFAR6 promoter showed that the AtFAR6 gene is expressed in various tissues of the plant with a distinct pattern compared to that of other Arabidopsis FARs, suggesting specialized functions in planta.
Assuntos
Aldeído Oxirredutases/biossíntese , Proteínas de Arabidopsis/biossíntese , Arabidopsis/enzimologia , Proteínas de Cloroplastos/biossíntese , Cloroplastos/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Aldeído Oxirredutases/química , Aldeído Oxirredutases/genética , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Cloroplastos/química , Proteínas de Cloroplastos/genética , Cloroplastos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato/fisiologia , Nicotiana/enzimologia , Nicotiana/genéticaRESUMO
Fatty alcohols and derivatives are important for proper deposition of a functional pollen wall. Mutations in specific genes encoding fatty acid reductases (FAR) responsible for fatty alcohol production cause abnormal development of pollen. A disrupted AtFAR2 (MS2) gene in Arabidopsis thaliana results in pollen developing an abnormal exine layer and a reduced fertility phenotype. AtFAR2 has been shown to be targeted to chloroplasts and in a purified form to be specific for acyl-ACP substrates. Here, we present data on the in vitro and in planta characterizations of AtFAR2 from A. thaliana and show that this enzyme has the ability to use both, C16:0-ACP and C16:0-CoA, as substrates to produce C16:0-alcohol. Our results further show that AtFAR2 is highly similar in properties and substrate specificity to AtFAR6 for which in vitro data has been published, and which is also a chloroplast localized enzyme. This suggests that although AtFAR2 is the major enzyme responsible for exine layer functionality, AtFAR6 might provide functional redundancy to AtFAR2.
Assuntos
Acil Coenzima A/química , Aldeído Oxirredutases/química , Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Proteína de Transporte de Acila/química , Aldeído Oxirredutases/biossíntese , Aldeído Oxirredutases/genética , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/genética , Álcoois Graxos/química , Álcoois Graxos/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Folhas de Planta/enzimologia , Soroalbumina Bovina , Especificidade por Substrato , NicotianaRESUMO
The reduction of acyl-CoA or acyl-ACP to fatty alcohol occurs via a fatty aldehyde intermediate. In prokaryotes this reaction is thought to be performed by separate enzymes for each reduction step while in eukaryotes these reactions are performed by a single enzyme without the release of the intermediate fatty aldehyde. However, here we report that a purified fatty acyl reductase from Marinobacter aquaeolei VT8, evolutionarily related to the fatty acyl reductases in eukaryotes, catalysed both reduction steps. Thus, there are at least two pathways existing among prokaryotes for the reduction of activated acyl substrates to fatty alcohol. The Marinobacter fatty acyl reductase studied has a wide substrate range in comparison to what can be found among enzymes so far studied in eukaryotes.
Assuntos
Acil Coenzima A/metabolismo , Aldeído Oxirredutases/metabolismo , Proteínas de Bactérias/metabolismo , Álcoois Graxos/metabolismo , Marinobacter/enzimologia , Aldeído Oxirredutases/química , Proteínas de Bactérias/química , Marinobacter/metabolismo , Oxirredução , FilogeniaRESUMO
Very long chain primary alcohols are significant components in cuticle waxes of plants. Fatty acyl-CoA reductases (FARs) catalyze the formation of a fatty alcohol from an acyl-CoA. The Arabidopsis (Arabidopsis thaliana) genome contains eight genes homologous to FAR genes from jojoba (Simmondsia chinensis), silk moth, wheat and mouse. Expression of six Arabidopsis FAR homologs in Escherichia coli resulted in production of alcohols from endogenous E. coli fatty acids by five of these genes, confirming that they encode for FAR enzymes. Only a truncated splicing version of the sixth gene was found, and this gene yielded a protein with no FAR activity. The five functional FAR enzymes yielded distinctly different compositions of fatty alcohols when expressed in E. coli, indicating that the different enzymes may be involved in the production of different types of alcohols in plant cells.