Rasyonel ve evolusyoner enzim dizaynı yolu ile iki (ß?)8 ?barrel enziminin katalitik aktivitelerinin değiştirilmesi
Küçük Resim Yok
Tarih
2009
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
TIM fıçı olarak da bilinen (ß?)8-fıçı, enzimlerin fonksiyon, stabilite ve evrimsel özelliklerinin anlaşılmasında mükemmel bir modeldir. Triptofan biyosentez yolundaki üç TIM fıçı, yapısal benzerlikleri ve korunmuş fosfat bağlanma bölgeleri nedeniyle ıraksak evrimle oluşmuş enzimlere en iyi örneklerdir. Yapısal benzerliklere dayanarak fosforibozil antranilat izomeraz (TrpF) ve bifonksiyonel triptofan sentaz ?-altbiriminin (TrpA) gen duplikasyonu ve farklılaşma sonucu ortak bir öncüden evrimleştikleri öne sürülmüştür.Bu çalışmada TrpA'ya TrpF aktivitesi kazandırılması için yönlendirilmiş evrim yaklaşımı kullanıldı. trpA geninde error-prone PCR veya DNA shuffling yöntemleri kullanılarak hazırlanan gen kütüphanelerinden in vivo komplementasyon yoluyla TrpF aktivitesine sahip varyantlar seçildi ve in vitro TrpF aktiviteleri belirlendi. TrpF'ye TrpA aktivitesi kazandırılması için rasyonel dizayn yoluyla belirlenen amino asit değişiklikleri gerçekleştirildi ve TrpF varyantının TrpA aktivitesi kazanıp kazanmadığı araştırıldı
The (ß?)8-barrel, which is also known as TIM barrel, is an excellent model to investigate the functional, stability, and the evolutionary properties of enzymes. Three TIM barrels in the tryptophan biosynthetic pathway are the clearest examples of divergently related enzymes because of the structural similarities and conserved phosphate binding sites. Based on the structural similarities, it has been postulated that phosphoribosylanthranilate isomerase and the ?-subunit of the bifunctional tryptophan synthase are descendants of a common ancestor from which they diverged by gene duplication and diversification.In this study, we used the directed evolution approach in order to establish TrpF activity on the protein scaffold of TrpA. In vivo complementation was used to select the variants with TrpF activity from the gene libraries, which were generated using error-prone PCR or DNA shuffling. In vitro TrpF activities of the variants were measured. For the establishment of TrpA activity on TrpF, the amino acid exchanges which were obtained from the rational design, were introduced. It was investigated whether TrpF variant gained TrpA activity.
The (ß?)8-barrel, which is also known as TIM barrel, is an excellent model to investigate the functional, stability, and the evolutionary properties of enzymes. Three TIM barrels in the tryptophan biosynthetic pathway are the clearest examples of divergently related enzymes because of the structural similarities and conserved phosphate binding sites. Based on the structural similarities, it has been postulated that phosphoribosylanthranilate isomerase and the ?-subunit of the bifunctional tryptophan synthase are descendants of a common ancestor from which they diverged by gene duplication and diversification.In this study, we used the directed evolution approach in order to establish TrpF activity on the protein scaffold of TrpA. In vivo complementation was used to select the variants with TrpF activity from the gene libraries, which were generated using error-prone PCR or DNA shuffling. In vitro TrpF activities of the variants were measured. For the establishment of TrpA activity on TrpF, the amino acid exchanges which were obtained from the rational design, were introduced. It was investigated whether TrpF variant gained TrpA activity.
Açıklama
Anahtar Kelimeler
Biyokimya, Biochemistry