Mimicking enzyme evolution by generating new (betaalpha)8-barrels from (betaalpha)4-half-barrels.
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 (betaalpha)8-barrel, which is the most frequent fold among enzymes, has evolved by the duplication, fusion, and mixing of (betaalpha)4-half-barrel domains. Here, we mimicked this evolutionary strategy by generating in vitro (betaalpha)8-barrels from (betaalpha)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 (betaalpha)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 beta-barrel of HisF-CC, the monomeric and compact (betaalpha)8-barrel protein HisF-C*C was generated. Moreover, the genes for the N- and C-terminal (betaalpha)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 (betaalpha)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 (betaalpha)8-barrels with novel functions might have evolved by the exchange of (betaalpha)4-half-barrel domains with distinct functional properties.
Höcker
Birte
B
Institut für Biochemie, Universität zu Köln, Otto-Fischer-Strasse 12-14, D-50674 Köln, Germany.
Claren
Jörg
J
Sterner
Reinhard
R
D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000619:Aminohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D019143:Evolution, Molecular; D020440:Gene Duplication; D005798:Genes, Bacterial; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020124:Thermotoga maritima / Q000201:enzymology / Q000235:genetics
D004269:DNA, Bacterial; D011994:Recombinant Proteins; C087227:imidazole glycerol phosphate synthase; D000619:Aminohydrolases; D019747:Aldose-Ketose Isomerases; C079656:Phosphoribosyl-5-amino-1-phosphoribosyl-4-imidazolecarboxiamide isomerase