Site‐directed mutagenesis of maize recombinant C4‐pyruvate,orthophosphate dikinase at the phosphorylatable target threonine residue

FEBS Letters

Lee, Mary E.²  Moorman, Molly A.²  Chollet, Raymond¹  Shameekumar, Patil¹  Chastain, Chris J.² 

[1] Department of Biochemistry, University of Nebraska-Lincoln, G.W. Beadle Center, Lincoln, NE 68588-0664, USA;Department of Biology, Moorhead State University, Moorhead, MN 56563, USA

关键词: Pyruvate;    Orthophosphate dikinase;    Regulatory protein;    C4 plant;    C4 photosynthesis;    Maize;    Zea mays;    DTT;    dithiothreitol;    IPTG;    isopropyl-β-thiogalactoside;    PEP;    phosphoenolpyruvate;    PMSF;    phenylmethylsulfonyl fluoride;    PPDK;    pyruvate;    orthophosphate dikinase;    RP;    regulatory protein;   

DOI  :  10.1016/S0014-5793(97)00884-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A key regulatory enzyme of the C₄-photosynthetic pathway is stromal pyruvate,orthophosphate dikinase (PPDK, EC 2.7.9.1). As a pivotal enzyme in the C₄ pathway, it undergoes diurnal light–dark regulation of activity which is mediated by a single bifunctional regulatory protein (RP). RP specifically inactivates PPDK in the dark by an ADP-dependent phosphorylation of an active-site Thr residue (Thr-456 in maize). Conversely, RP activates inactive PPDK in the light by phosphorolytic dephosphorylation of this target Thr-P residue. We have employed a His-tagged maize recombinant C₄ PPDK for directed mutagenesis of this active-site regulatory Thr. Three such mutants (T456V, T456S, T456D) were analyzed with respect to overall catalysis and regulation by exogenous maize RP. Substitution with Val and Ser at this position does not affect overall catalysis, whereas Asp abolishes enzyme activity. With respect to regulation by RP, it was found that Ser can effectively substitute for the wild-type Thr residue in that mutant enzyme is phosphorylated and inactivated by RP. The T456V mutant, however, could not be phosphorylated and was, thus, resistant to ADP-dependent inactivation by RP.

【 授权许可】

Unknown