Enzyme evolution [R. Sterner]

It is plausible to assume that the highly efficient and specific contemporary enzymes have evolved from less sophisticated precursors.

We are studying this process for the (ba)₈-barrels enzymes ProFAR isomerase (HisA) and imidazole glycerol phosphate synthase (HisF). They possess a striking internal two-fold symmetry. The pairs of N-terminal halves (designated HisA-N and HisF-N), which consist of the first four (ba) units, and the pairs of C-terminal halves (designated HisA-C and HisF-C), which consist of the last four (ba) units, display sequence identities between 16 and 26 % and rmsd values of their main-chain non-hydrogen atoms between 1.4 and 2.1 Å. When produced separately, the half-barrels HisF-N and HisF-C are homodimeric proteins with native secondary and tertiary structures, but without measurable catalytic activity. When co-expressed in vivo or refolded together in vitro, the two proteins assemble to a catalytically fully active HisF-NC heterodimer. It was concluded that both HisF and HisA are composed of two structural domains, namely the corresponding N- and C-terminal half-barrels.

These results suggest an evolutionary scenario according to which a primordial gene encoding a (ba)₄-half-barrel as subunit of a homodimeric enzyme was duplicated and fused to yield a monomeric, ancestral (ba)₈-barrel, which might have been the precursor of contemporary (ba)₈-barrel enzymes. These postulated evolutionary events were reconstructed experimentally by generating new (ba)₈-barrels from existing (ba)₄-half-barrels. To this end, HisF-C was duplicated, fused and optimised to yield the stable and monomeric HisF-C***C barrel, whose X-ray structure could be solved at high resolution. Moreover, the N- and C-terminal half-barrels of HisA and HisF were fused crosswise to yield the chimeric HisAF and HisFA proteins. Using a combination of random mutagenesis and selection in vivo, high catalytic activity was established on the HisAF scaffold. The results show that stable and catalytically active (ba)₈-barrels can be assembled in the laboratory by fusing, mixing and matching of (ba)₄-half-barrels. Similar events might have occurred in the course of natural evolution.