In vitro heat effect on heterooligomeric subunit assembly of thermostable indolepyruvate ferredoxin oxidoreductase

Indolepyruvate ferredoxin oxidoreductase (IOR) from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 catalyzes the oxidative decarboxylation of arylpyruvates by forming a heterooligomeric complex (alpha2beta2). The genes iorA and iorB which encode respective alpha and beta 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 alpha subunit nor beta subunit. The alpha and beta 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 alpha and beta subunits did not possess any IOR activity even though the mixed sample was heat treated again. IOR alpha and beta 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 alpha and beta were converted to an active high molecular weight complex (195 kDa) which corresponds to the alpha2beta2 structure. However, the active complex was not formed without heat treatment, suggesting that high temperature environments are important for the heterooligomerization of IOR subunits.