Dissection of a (beta alpha)(8)-barrel enzyme into two folded halves

The (beta alpha)(8)-barrel, which is the most frequently encountered protei n fold, is generally considered to consist of a single structural domain. H owever, the X-ray structure of the imidazoleglycerol phosphate synthase (Hi sF) from Thermotoga maritima has identified it as a (beta alpha)(8)-barrel made up of two superimposable subdomains (HisF-N and HisF-C). HisF-N consis ts of the four N-terminal (beta alpha) units and HisF-C of the four C-termi nal (beta alpha) units. It has been postulated, therefore, that HisF evolve d by tandem duplication and fusion from an ancestral half-barrel. To test t his hypothesis, HisF-N and HisF-C were produced in Escherichia coli, purifi ed and characterized. Separately, HisF-N and HisF-C are folded proteins, bu t are catalytically inactive. Upon coexpression in vivo or joint refolding in vitro, HisF-N and HisF-C assemble to the stoichiometric and catalyticall y fully active HisF-NC complex. These findings support the hypothesis that the (beta alpha)(8)-barrel of HisF evolved from an ancestral half-barrel an d have implications for the folding mechanism of the members of this large protein family.