1 非天然型及び天然型アミノ酸部位特異的導入実験に基づくスクアレン環化機構の考察 : カチオン/π相互作用とアミノ酸側鎖のバルクサイズの重要性(口頭発表の部)

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The enzymatic cyclisations of acyclic squalene 1 and 2,3-oxidosqualene (OS) to form polycyclic triterpenes have fascinated chemists and biochemists for over half a century. X-ray crystallographic analysis of prokaryotic squalene-hopene cyclase (SHC) was reported in 1997. More recently, three-dimensional structure of human lanosterol synthase has successfully been elucidated. SHC catalyses the conversion of 1 into the pentacyclic triterpenes of hopene 2 and hopanol 3, the ratio being ca. 5: 1, respectively. This polycyclization cascade is attained by single enzyme. 1 is folded in all pre-chair conformation inside the reaction cavity and cyclized in regio- and stereospecific manner through a series of carbocationic intermediates, leading to the formation of new five C-C bonds and nine chiral centers. 1. Critical role of steric bulk size of amino acid residues for the folding conformation of squalene. Several examples are presented in this symposium. A306V mutant gave the tetracyclic products that are constructed by the chair-chair-chair-boat conformation. The Y420W and L607W afforded a novel enzymic product, neoachillapentaene, which is produced via the constrained boat form. These single amino acid substituted mutants enforced 1 into a boat structure, despite all pre-chair conformation being adopted by the wild-type SHC. These findings indicate that the folding conformation is directed by the steric bulk size of the active site residues. 2. Eukaryotic cyclases (OSCs) accept only (3S)-2,3-oxidosqualene (OS) as the substrate, but are inert to the (3R)-OS. The original substrate of SHC is 1, but both (3S)- and (3R)-OSs also can be accepted to afford 3β-hydroxy- and 3α-hydroxyhopenes, respectively. Comparison of amino acid alignment between OSCs and SHCs indicates that Gly600 is conserved in all SHCs, but this residue is lacking in all the OSCs. The G600-deletion mutant was constructed and incubated with 1 and the (3R,S)-racemic OSs. This mutant accepted only (3S)-OS, but were inert to 1 and (3R)-OS, indicating that the substrate specificity of prokaryotic cyclase was successfully altered to that of eukaryotic cyclases. 3. The site-specific incorporation experiments of unnatural amino acids into SHC were planned to validate the concept of cation/π interaction for the polycyclisation cascade of squalene. The targeted positions of SHC were mutated into amber codon (UAG). The site-specific in vivo incorporation of O-methoxy- and p-aminophenylalanines into positions 305 and 605 was successfully achieved according to Schultz's protocol. By using the system of cell-free protein synthesis, fluorinated phenylalanines were site-specifically incorporated into their positions. The specific activities of the SHCs into that fluorophenylalanines were site-specifically incorporated were decreased in inverse proportion to the number of substituted fluorine atoms, clearly indicating that cation/π interaction operated during the polycyclisation cascade.
天然有機化合物討論会の論文
2005-09-15

1 非天然型及び天然型アミノ酸部位特異的導入実験に基づくスクアレン環化機構の考察 : カチオン/π相互作用とアミノ酸側鎖のバルクサイズの重要性(口頭発表の部)

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