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In vitro heat effect on heterooligomeric subunit assembly of thermostable indolepyruvate ferredoxin oxidoreductase
Author(s) -
Siddiqui Masood A,
Fujiwara Shinsuke,
Takagi Masahiro,
Imanaka Tadayuki
Publication year - 1998
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)00998-3
Subject(s) - protein subunit , biochemistry , oxidoreductase , hyperthermophile , ferredoxin , chemistry , escherichia coli , g alpha subunit , biology , microbiology and biotechnology , enzyme , gene , archaea
Indolepyruvate ferredoxin oxidoreductase (IOR) from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 catalyzes the oxidative decarboxylation of arylpyruvates by forming a heterooligomeric complex (α 2 β 2 ). The genes iorA and iorB which encode respective α and β subunits, were coexpressed heterologously in Escherichia coli cells under anaerobic conditions. IOR activity was detected from the cell extract containing both subunits and its activity was enhanced by in vitro heat treatment prior to the assay. The iorA and iorB were expressed individually and each subunit was examined for enzymatic activity with and without heat treatment. IOR activity was detected neither from the extract of α subunit nor β subunit. The α and β subunits were mixed and then IOR activity was examined. Weak IOR activity was detected without heat treatment, however, upon heat treatment its activity was enhanced. The mixture of individually heat treated α and β subunits did not possess any IOR activity even though the mixed sample was heat treated again. IOR α and β subunits were individually purified to homogeneity, mixed with or without heat treatment and subunit assembly was examined by determining molecular mass. Upon heat treatment, inactive α and β were converted to an active high molecular weight complex (195 kDa) which corresponds to the α 2 β 2 structure. However, the active complex was not formed without heat treatment, suggesting that high temperature environments are important for the hetero‐oligomerization of IOR subunits.