Purification, molecular cloning, and sequence analysis of sucrose-6F-phosphate phosphohydrolase from plants.
Sucrose-6(F)-phosphate phosphohydrolase (SPP; EC ) catalyzes the final step in the pathway of sucrose biosynthesis and is the only enzyme of photosynthetic carbon assimilation for which the gene has not been identified. The enzyme was purified to homogeneity from rice (Oryza sativa L.) leaves and partially sequenced. The rice leaf enzyme is a dimer with a native molecular mass of 100 kDa and a subunit molecular mass of 50 kDa. The enzyme is highly specific for sucrose 6(F)-phosphate with a K(m) of 65 microM and a specific activity of 1250 micromol min(-1) mg(-1) protein. The activity is dependent on Mg(2+) with a remarkably low K(a) of 8-9 microM and is weakly inhibited by sucrose. Three peptides from cleavage of the purified rice SPP with endoproteinase Lys-C showed similarity to the deduced amino acid sequences of three predicted open reading frames (ORF) in the Arabidopsis thaliana genome and one in the genome of the cyanobacterium Synechocystis sp. PCC6803, as well as cDNA clones from Arabidopsis, maize, and other species in the GenBank database of expressed sequence tags. The putative maize SPP cDNA clone contained an ORF encoding a 420-amino acid polypeptide. Heterologous expression in Escherichia coli showed that this cDNA clone encoded a functional SPP enzyme. The 260-amino acid N-terminal catalytic domain of the maize SPP is homologous to the C-terminal region of sucrose-phosphate synthase. A PSI-BLAST search of the GenBank database indicated that the maize SPP is a member of the haloacid dehalogenase hydrolase/phosphatase superfamily.
Lunn
J E
JE
Commonwealth Scientific and Industrial Research Organization Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia. john.lunn@pi.csiro.au
Ashton
A R
AR
Hatch
M D
MD
Heldt
H W
HW
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018744:DNA, Plant; D004926:Escherichia coli; D005636:Fructosephosphates / Q000378:metabolism; D015870:Gene Expression; D017343:Genes, Plant; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology / Q000235:genetics; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein / Q000379:methods; D017386:Sequence Homology, Amino Acid; D003313:Zea mays / Q000201:enzymology / Q000235:genetics
D018744:DNA, Plant; D005636:Fructosephosphates; C027618:fructose-6-phosphate; D010744:Phosphoric Monoester Hydrolases; C022697:sucrose-phosphatase