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

Abstract

The (beta alpha)(8)-barrel, which is the most frequently encountered protein fold, is generally considered to consist of a single structural domain. However, the X-ray structure of the imidazoleglycerol phosphate synthase (HisF) 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 consists of the four N-terminal (beta alpha) units and HisF-C of the four C-terminal (beta alpha) units. It has been postulated, therefore, that HisF evolved by tandem duplication and fusion from an ancestral half-barrel. To test this hypothesis, HisF-N and HisF-C were produced in Escherichia coli, purified and characterized. Separately, HisF-N and HisF-C are folded proteins, but are catalytically inactive. Upon coexpression in vivo or joint refolding in vitro, HisF-N and HisF-C assemble to the stoichiometric and catalytically fully active HisF-NC complex. These findings support the hypothesis that the (beta alpha)(8)-barrel of HisF evolved from an ancestral half-barrel and have implications for the folding mechanism of the members of this large protein family.