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Site‐directed mutagenesis of serine 158 demonstrates its role in spinach leaf sucrose‐phosphate synthase modulation
Author(s) -
Toroser Dikran,
McMichael Robert,
Krause KlausePeter,
Kurreck Jens,
Sonnewald Uwe,
Stitt Mark,
Huber Steven C.
Publication year - 1999
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1999.00389.x
Subject(s) - spinach , biochemistry , sucrose phosphate synthase , enzyme , mutant , biology , microbiology and biotechnology , phosphorylation , mutagenesis , enzyme assay , transgene , wild type , chemistry , sucrose synthase , gene , invertase
Summary Site‐directed mutagenesis of spinach sucrose‐phosphate synthase (SPS) was performed to investigate the role of Ser158 in the modulation of spinach leaf SPS. Tobacco plants expressing the spinach wild‐type (WT), S158A, S158T and S157F/S158E SPS transgenes were produced. Expression of transgenes appeared not to reduce expression of the tobacco host SPS. SPS activity in the WT and the S158T SPS transgenics showed light/dark modulation, whereas the S158A and S157F/S158E mutants were not similarly light/dark modulated: the S158A mutant enzyme was not inactivated in the dark, and the S157F/S158E was not activated in the light. The inability to modulate the activity of the S158A mutant enzyme by protein phosphorylation was demonstratedin vitro. The WT spinach enzyme immunopurified from dark transgenic tobacco leaves had a low initial activation state, and could be activated by PP2A and subsequently inactivated by SPS‐kinase plus ATP. Rapid purification of the S158A mutant enzyme from dark leaves of transgenic plants using spinach‐specific monoclonal antibodies yielded enzyme that had a high initial activation state, and pre‐incubation with leaf PP2A or ATP plus SPS‐kinase (the PK III enzyme) caused little modulation of activity. The results demonstrate the regulatory significance of Ser158 as the major site responsible for dark inactivation of spinach SPSin vivo, and indicate that the significance of phosphorylation is the introduction of a negative charge at the Ser158 position.