Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Development of the feedback resistant pheAFBR from E. coli and studies on its biochemical characteristics

E. coli 유래 pheA 유전자의 되먹임제어 저항성 돌연변이의 구축과 그 단백질의 생화학적 특성 연구

  • Cao, Thinh-Phat (Department of Cellular and Molecular Medicine, Chosun University School of Medicien) ;
  • Lee, Sang-Hyun (Department of Biotechnology, Graduate School, Korea University) ;
  • Hong, KwangWon (Department of Food Science and Biotechnology, Dongguk University-Seoul) ;
  • Lee, Sung Haeng (Department of Cellular and Molecular Medicine, Chosun University School of Medicien)
  • 카오틴팟 (조선대학교 의과대학 세포분자의과학교실) ;
  • 이상현 (고려대학교 대학원 생명공학과) ;
  • 홍광원 (동국대학교 식품생명공학과) ;
  • 이성행 (조선대학교 의과대학 세포분자의과학교실)
  • Received : 2016.09.01
  • Accepted : 2016.09.23
  • Published : 2016.09.30
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Abstract

The bifunctional PheA protein, having chorismate mutase and prephenate dehydratase (CMPD) activities, is one of the key regulatory enzymes in the aromatic amino acid biosynthesis in Escherichia coli, and is negatively regulated by an end-product, phenyalanine. Therefore, PheA protein has been thought as useful for protein engineering to utilize mass production of essential amino acid phenylalanine. To obtain feedback resistant PheA protein against phenylalanine, we mutated by using random mutagenesis, extensively screened, and obtained $pheA^{FBR}$ gene encoding a feedback resistant PheA protein. The mutant PheA protein contains substitution of Leu to Phe at the position of 118, displaying that higher affinity (about $290{\mu}M$) for prephenate in comparison with that (about $850{\mu}M$) of wild type PheA protein. Kinetic analysis showed that the saturation curve of $PheA^{FBR}$ against phenyalanine is hyperbolic rather than that of $PheA^{WT}$, which is sigmoidal, indicating that the L118F mutant enzyme has no cooperative effects in prephenate binding in the presence of phenylalanine. In vitro enzymatic assay showed that the mutant protein exhibited increased activity by above 3.5 folds compared to the wild type enzyme. Moreover, L118F mutant protein appeared insensitive to feedback inhibition with keeping 40% of enzymatic activity even in the presence of 10 mM phenylalanine at which the activity of wild type $PheA^{WT}$ was not observed. The substitution of Leu to Phe in CMPD may induce significant conformational change for this enzyme to acquire feedback resistance to end-product of the pathway by modulating kinetic properties.

E. coli의 PheA 단백질은 chorismate mutase and prephenate dehydratase (CMPD) 활성을 가지며 마지막 산물인 페닐알라닌에 의하여 되먹임제어가 되는 생합성 경로의 주요 조절 효소 중의 하나이다. 그러므로, 이 PheA 단백질은 필수 아미노산 중의 하나인 페닐알라닌의 대량 생산에 이용하기 위한 단백질 공학의 타겟이 될 수 있다. 이러한 목적으로 PheA 단백질의 마지막 생산물인 페닐알라닌에 의한 되먹임저해 저항성 유전자원을 선별하였다. 이 유전자의 산물인 $PheA^{FBR}$은 118번째 류신이 페닐알라닌으로 치환되었고, 기질인 prephenate에 대한 친화도가 야생주단백질과 비교하여 약 3.5배 정도 높았다. $PheA^{FBR}$은 세포내에서 축척되어져 되먹임저해를 하는 페닐알라닌 농도에서(약1 mM와 10 mM)에서도 50%와 40%의 활성을 유지 하고 있었고, 페닐알라닌 존재하에서 기질의 결합 성향이 협동적(cooperative) 모드에서 단독적(hyperbolic) 모드로 전환되었다. 이는 기존 연구와 비교해 볼 때, 이 돌연변이 부위는 이 융합기능 효소인 PheA 단백질의 새로운 조절 부위의 존재를 암시 한다. 효소 동력학적 결과는 PheA 단백질의 되먹임저해 저항성 획득이 아미노산 돌연변이에 의한 단백질 구조의 변화 유도에 의한 것으로 생각된다. 더 나아가, 본 연구에서 선별된 돌연변이 유전자는 생물전환법을 이용한 필수아미노산 생산에 산업적으로 응용 가능성이 있다.

Keywords

References

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