Isolation and characterization of a higher plant ADP‐glucose pyrophosphorylase small subunit homotetramer

FEBS Letters

Kavakli, Ibrahim H.¹  Salamone, Peter R.¹  Greene, Thomas W.²  Okita, Thomas W.¹ 

[1] Institute for Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA;Dow AgroSciences LLC, 9330 Zionsville Rd, Indianapolis, IN 46268, USA

关键词: ADP-glucose pyrophosphorylase;    Mutagenesis;    Starch;    Homotetramer;    Allosteric regulation;    AGPase;    adenosine 5′-diphosphate glucose pyrophosphorylase;    3-PGA;    3-phosphoglycerate;    Pi;    inorganic phosphate;    PPi;    inorganic pyrophosphate;    WT;    wild type;    SS;    small subunit;    LS;    large subunit;    PMSF;    phenylmethylsulfonyl fluoride;   

DOI  :  10.1016/S0014-5793(00)01985-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

ADP-glucose pyrophosphorylase (AGPase) is the allosterically regulated gateway for carbon entry into transient and storage starch in plants as well as glycogen in bacteria. This enzyme plays a key role in the modulation of photosynthetic efficiency in source tissues and directly determines the level of storage starch in sink tissues, thus influencing overall crop yield potential. AGPase is a tetrameric enzyme; in higher plants it consists of two regulatory large subunits (LS) and two catalytic small subunits (SS), while in cyanobacteria and prokaryotes the enzyme is homotetrameric. The potato SS gene in pML10 was mutated by hydroxylamine and mutants were screened for elevated homotetrameric activity by iodine vapor staining. This search strategy led to the isolation of SS mutants (SUP-1, TG-15) that had pyrophosphorylase activity in the absence of the LS. TG-15 has a leucine to phenylalanine change at position 48 (L₄₈F) that corresponds to a phenylalanine residue at the analogous position in the Escherichia coli homotetrameric AGPase as well as a valine to isoleucine change at position 59 (V₅₉I). TG-15 was partially purified and kinetic analysis revealed substrate and effector affinities equal to wild type heterotetrameric enzyme with the exception of ATP binding.

【 授权许可】

Unknown