RESUMO
Crystal structures of peroxisomal Arabidopsis thaliana 3-ketoacyl-CoA thiolase (AtKAT), an enzyme of fatty acid beta-oxidation, are reported. The subunit, a typical thiolase, is a combination of two similar alpha/beta domains capped with a loop domain. The comparison of AtKAT with the Saccharomyces cerevisiae homologue (ScKAT) structure reveals a different placement of subunits within the functional dimers and that a polypeptide segment forming an extended loop around the open catalytic pocket of ScKAT converts to alpha-helix in AtKAT, and occludes the active site. A disulfide is formed between Cys192, on this helix, and Cys138, a catalytic residue. Access to Cys138 is determined by the structure of this polypeptide segment. AtKAT represents an oxidized, previously unknown inactive form, whilst ScKAT is the reduced and active enzyme. A high level of sequence conservation is observed, including Cys192, in eukaryotic peroxisomal, but not mitochondrial or prokaryotic KAT sequences, for this labile loop/helix segment. This indicates that KAT activity in peroxisomes is influenced by a disulfide/dithiol change linking fatty acid beta-oxidation with redox regulation.
Assuntos
Acetil-CoA C-Aciltransferase/química , Proteínas de Arabidopsis/química , Ácidos Graxos/química , Peroxissomos/metabolismo , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Acetil-CoA C-Aciltransferase/genética , Acetil-CoA C-Aciltransferase/metabolismo , Sequência de Aminoácidos , Animais , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de SequênciaRESUMO
The enzyme 3-ketoacyl-CoA thiolase (KAT) (EC 2.3.1.16) catalyses a key step in fatty acid beta-oxidation. In Arabidopsis thaliana, expression of the KAT2 gene is known to be required for the efficient mobilization of triacylglycerol during germination and seedling establishment. Here, data from the Arabidopsis kat2-1 mutant are presented, showing that perturbation of beta-oxidation also affects vegetative growth and reproductive success. In the wild type, the KAT2 protein was detected in all organs tested. In the kat2-1 mutant, rosette leaf area and dry weight, but not leaf number, were greatly increased relative to wild type. Global proliferative arrest of flowering was delayed, resulting in increased silique production in kat2-1 plants. However, total silique dry weight was not increased. kat2-1 siliques were smaller and had a reduced seed number caused by increased ovule abortion. In kat2-1 ovules, carbon flow into sugars via gluconeogeneis and respiration were both reduced in comparison to the wild type. In conclusion, these data indicate that a functional beta-oxidation pathway is required to maintain the balance between silique development and the continued initiation of floral meristems.
Assuntos
Acetil-CoA C-Aciltransferase/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Carbono/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Mutação , Fenótipo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Reprodução/genéticaRESUMO
A barley (Hordeum vulgare L.) cDNA, PM19, encoding a putative plasma membrane protein was isolated through differential screening of a dormant wild oat embryo library. PM19 is expressed in barley embryos from mid-embryogenesis up to maturity. PM19 mRNA levels decline upon germination, whereas dormant embryos retained high levels of message for up to 72 h of imbibition. PM19 mRNA levels also remained high or were reinduced in non-dormant embryos by treatments that prevented germination (250 mm NaCl, 10% sorbitol, or 50 microm ABA). The PM19 protein sequence is highly conserved in monocotyledonous and dicotyledonous plants.
Assuntos
Hordeum/genética , Proteínas de Membrana/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Membrana Celular/genética , Membrana Celular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Germinação/efeitos dos fármacos , Germinação/genética , Hordeum/metabolismo , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sementes/genética , Sementes/metabolismo , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/farmacologia , Sorbitol/farmacologiaRESUMO
The barley protein limit dextrinase inhibitor (LDI), structurally related to the alpha-amylase/trypsin inhibitor family, is an inhibitor of the starch debranching enzyme limit dextrinase (LD). In order to investigate the function of LDI, and the consequences for starch metabolism of reduced LDI activity, transgenic barley plants designed to downregulate LDI by antisense were generated. Homozygous antisense lines with reduced LDI protein level and activity were analysed and found to have enhanced free LD activity in both developing and germinating grains. In addition the antisense lines showed unpredicted pleiotropic effects on numerous enzyme activities, for example, alpha- and beta-amylases and starch synthases. Analysis of the starch showed much reduced numbers of the small B-type starch granules, as well as reduced amylose relative to amylopectin levels and reduced total starch. The chain length distribution of the amylopectin was modified with less of the longer chains (>25 units) and enhanced number of medium chains (10-15 units). These results suggest an important role for LDI and LD during starch synthesis as well as during starch breakdown.