Open AccessPurification, molecular cloning, and sequence analysis of sucrose-6 F -phosphate phosphohydrolase from plants
Author(s) -
John E. Lunn,
Anthony R. Ashton,
M.D. Hatch,
Hans-Walter Heldt
Publication year - 2000
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.230430197
Subject(s) - biochemistry , biology , complementary dna , peptide sequence , sucrose phosphate synthase , amino acid , heterologous expression , open reading frame , molecular cloning , genbank , enzyme , microbiology and biotechnology , sucrose synthase , gene , recombinant dna , invertase
Sucrose-6F -phosphate phosphohydrolase (SPP; EC3.1.3.24 ) 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 6F -phosphate with aK m of 65 μM and a specific activity of 1250 μmol min−1 mg−1 protein. The activity is dependent on Mg2+ with a remarkably lowK a of 8–9 μM 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 theArabidopsis thaliana genome and one in the genome of the cyanobacteriumSynechocystis sp. PCC6803, as well as cDNA clones fromArabidopsis , 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 inEscherichia 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.