RESUMO
Ascorbate (vitamin C) is an essential antioxidant and enzyme cofactor in both plants and animals. Ascorbate concentration is tightly regulated in plants, partly to respond to stress. Here, we demonstrate that ascorbate concentrations are determined via the posttranscriptional repression of GDP-l-galactose phosphorylase (GGP), a major control enzyme in the ascorbate biosynthesis pathway. This regulation requires a cis-acting upstream open reading frame (uORF) that represses the translation of the downstream GGP open reading frame under high ascorbate concentration. Disruption of this uORF stops the ascorbate feedback regulation of translation and results in increased ascorbate concentrations in leaves. The uORF is predicted to initiate at a noncanonical codon (ACG rather than AUG) and encode a 60- to 65-residue peptide. Analysis of ribosome protection data from Arabidopsis thaliana showed colocation of high levels of ribosomes with both the uORF and the main coding sequence of GGP. Together, our data indicate that the noncanonical uORF is translated and encodes a peptide that functions in the ascorbate inhibition of translation. This posttranslational regulation of ascorbate is likely an ancient mechanism of control as the uORF is conserved in GGP genes from mosses to angiosperms.
Assuntos
Arabidopsis/genética , Ácido Ascórbico/biossíntese , Retroalimentação Fisiológica , Regulação da Expressão Gênica de Plantas , Fases de Leitura Aberta/genética , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Ácido Ascórbico/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Códon/genética , Regulação para Baixo/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos dos fármacos , Galactose/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Luciferases/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Fosfotransferases/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismoRESUMO
The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel beta-sheet gripping an alpha-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting beta-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S' side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp(32)-Asp(215) diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin.
Assuntos
Cucurbita/química , Proteínas de Plantas/química , Inibidores de Proteases/química , Sítios de Ligação , Cistatinas/química , Cistatinas/genética , Ressonância Magnética Nuclear Biomolecular , Pepsina A/química , Pepsina A/genética , Proteínas de Plantas/genética , Estrutura Secundária de Proteína , Homologia Estrutural de ProteínaRESUMO
The gene for one postulated enzyme that converts GDP-L-galactose to L-galactose-1-phosphate is unknown in the L-galactose pathway of ascorbic acid biosynthesis and a possible candidate identified through map-based cloning is the uncharacterized gene At4g26850. We identified a putative function for At4g26850 using PSI-Blast and motif searching to show it was a member of the histidine triad superfamily, which includes D-galactose uridyltransferase. We cloned and expressed this Arabidopsis gene and the homologous gene from Actinidia chinensis in Escherichia coli and assayed the expressed protein for activities related to converting GDP-L-galactose to L-galactose-1-P. The expressed protein is best described as a GDP-L-galactose-hexose-1-phosphate guanyltransferase (EC 2.7.7.), catalyzing the transfer of GMP from GDP-l-galactose to a hexose-1-P, most likely D-mannose-1-phosphate in vivo. Transient expression of this A. chinensis gene in tobacco leaves resulted in a >3-fold increase in leaf ascorbate as well as a 50-fold increase in GDP-L-galactose-D-mannose-1-phosphate guanyltransferase activity.
