Cloning, expression and characterization of a Nudix hydrolase that catalyzes the hydrolytic breakdown of ADP-glucose linked to starch biosynthesis in Arabidopsis thaliana.
'Nudix' hydrolases are widely distributed nucleotide pyrophosphatases that possess a conserved GX5EX7REUXEEXGU motif where U is usually isoleucine, leucine or valine. Among them, Escherichia coli ADP-sugar pyrophosphatase (ASPP) has been shown to catalyze the hydrolytic breakdown of ADP-glucose linked to bacterial glycogen biosynthesis. Comparisons of the 31 different Nudix-encoding sequences of the Arabidopsis genome with those coding for known bacterial and mammalian ASPPs identified one sequence possessing important divergences in the Nudix motif that, once expressed in E. coli, produced a protein with ASPP activity. This protein, designated as AtASPP, shares strong homology with hypothetical rice and potato proteins, indicating that ASPPs are widely distributed in both mono- and dicotyledonous plants. As a first step to test the possible involvement of plant ASPPs in regulating the intracellular levels of ADP-glucose linked to starch biosynthesis, we produced and characterized AtASPP-overexpressing Arabidopsis plants. Source leaves from these plants exhibited a large reduction in the levels of both ADP-glucose and starch, indicating that plant ASPPs catalyze the hydrolytic breakdown of a sizable pool of ADP-glucose linked to starch biosynthesis. No pleiotropic changes in maximum catalytic activities of enzymes closely linked to starch metabolism could be detected in AtASPP-overexpressing leaves. The overall information provides the first evidence for the existence of plant Nudix hydrolases that have access to an intracellular pool of ADP-glucose linked to starch biosynthesis.
Muñoz
Francisco José
FJ
Instituto de Agrobiotecnología, Universidad Pública de Navarra/Consejo Superior de Investigaciones Científicas/Gobierno de Navarra, Ctra. Mutilva s/n, 31192 Mutilva Baja, Navarra, Spain. francisco.munoz@unavarra.es
Baroja-Fernández
Edurne
E
Morán-Zorzano
María Teresa
MT
Alonso-Casajús
Nora
N
Pozueta-Romero
Javier
J
D000245:Adenosine Diphosphate Glucose / Q000378:metabolism*; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000502:physiology; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics; D051599:Glucose-1-Phosphate Adenylyltransferase / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000378:metabolism; D011755:Pyrophosphatases / Q000235:genetics* / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics; D013213:Starch / Q000378:metabolism*; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D000096964:Nudix Hydrolases
D000245:Adenosine Diphosphate Glucose; D029681:Arabidopsis Proteins; D029968:Escherichia coli Proteins; D051599:Glucose-1-Phosphate Adenylyltransferase; D011755:Pyrophosphatases; D011994:Recombinant Proteins; D013213:Starch; D013395:Sucrose; D000096964:Nudix Hydrolases