Mimicking enzyme evolution by generating new (βα) ₈ -barrels from (βα) ₄ -half-barrels

Abstract

Gene duplication and fusion events that multiply and link functional protein domains are crucial mechanisms of enzyme evolution. The analysis of amino acid sequences and three-dimensional structures suggested that the (␤␣) 8-barrel, which is the most frequent fold among enzymes, has evolved by the duplication, fusion, and mixing of (␤␣) 4-half-barrel domains. Here, we mimicked this evolutionary strategy by generating in vitro (␤␣) 8-barrels from (␤␣) 4-half-barrels that were deduced from the enzymes imidazole glycerol phosphate synthase (HisF) and N [(5 -phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide isomerase (HisA). To this end, the gene for the C-terminal (␤␣) 4-half-barrel (HisF-C) of HisF was duplicated and fused in tandem to yield HisF-CC, which is more stable than HisF-C. In the next step, by optimizing side-chain interactions within the center of the ␤-barrel of HisF-CC, the monomeric and compact (␤␣) 8-barrel protein HisF-C*C was generated. Moreover, the genes for the N-and C-terminal (␤␣) 4-half-barrels of HisF and HisA were fused crosswise to yield the chimeric proteins HisFA and HisAF. Whereas HisFA contains native secondary structure elements but adopts ill-defined association states, the (␤␣) 8-barrel HisAF is a stable and compact monomer that reversibly unfolds with high cooperativity. The results obtained suggest a previously undescribed dimension for the diversification of enzymatic activities: new (␤␣) 8-barrels with novel functions might have evolved by the exchange of (␤␣) 4-halfbarrel domains with distinct functional properties. chimeric proteins ͉ gene duplication ͉ histidine biosynthesis ͉ TIM-barrel

Potential of Fragment Recombination for Rational Design of Proteins

“…7−12 Diversification of (βα) 8 -barrel enzymes by exchange of "half-barrels" was further elucidated by combining halves from related (βα) 8 -barrel proteins. 8,13 In contrast, the flavodoxin-like fold is associated with a diverse mixture of functionalities such as response regulation in signaling systems, cofactor binding, or enzymatic activities. It is a three-layered fold made up of a parallel five-stranded β-sheet flanked by two α-helices on one side and three on the other, found both as an isolated domain and as part of multidomain proteins.…”

“…For the present study, protein fragments from two major folds were selected: the TIM-or (βα) 8 -barrel and the flavodoxin-like fold. The (βα) 8 -barrel, commonly found among enzymes, consists of a closed eight-stranded parallel β-sheet that forms a central barrel surrounded by eight α-helices.…”

“…The (βα) 8 -barrel, commonly found among enzymes, consists of a closed eight-stranded parallel β-sheet that forms a central barrel surrounded by eight α-helices. 6 The remarkable two-fold symmetry of two enzymes from histidine biosynthesis indicates that this fold evolved from an ancestral "half-barrel" fragment through duplication and fusion.…”

“…6 The remarkable two-fold symmetry of two enzymes from histidine biosynthesis indicates that this fold evolved from an ancestral "half-barrel" fragment through duplication and fusion. 7−12 Diversification of (βα) 8 -barrel enzymes by exchange of "half-barrels" was further elucidated by combining halves from related (βα) 8 -barrel proteins. 8,13 In contrast, the flavodoxin-like fold is associated with a diverse mixture of functionalities such as response regulation in signaling systems, cofactor binding, or enzymatic activities.…”

“…flavodoxin-like fold and the (βα) 8 -barrel are considered to belong to the nine most ancient protein folds. 14 We previously attempted to construct a chimeric (βα) 8 barrel by combining fragments from these two different folds.…”