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대장균 트립토판 생성효소의 소단위체간 상호조절

Intersubunit Communication of Escherichia coli Tryptophan Synthase

  • 조원진 (부산대학교 자연과학대학 분자생물학과) ;
  • 임운기 (부산대학교 자연과학대학 분자생물학과)
  • Cho, Won Jin (Department of Molecular Biology, College of Natural Sciences, Pusan National University) ;
  • Lim, Woon Ki (Department of Molecular Biology, College of Natural Sciences, Pusan National University)
  • 투고 : 2017.10.20
  • 심사 : 2017.11.16
  • 발행 : 2017.12.30

초록

대장균 트립토판 생성효소는 ${\alpha}_2{\beta}_2$ 복합체로 구성되며, 트립토판 생합성에서 최종 2 단계의 반응에 관여한다. 두 개의 소단위체는 분자 터널로 연결되어 있어, 기질 채널링이 일어난다. 활성 부위간 상호 조절하는 정교한 조절 기작에는 ${\alpha}$-루프 L6(${\alpha}L6$), ${\alpha}L2$, ${\alpha}L3$이 관여한다. 본 연구에서는 이 자리의 잔기치환체를 써서 소단위체에 특이적으로 결합하는 리간드의 영향을 조사하여 소단위체간 상호 조절기작에 따른 구조 변화를 살펴보았다. ${\alpha}TS$의 활성부위에 결합하는 D,L-${\alpha}$-glycerophosphate(GP)는 모든 잔기치환체를 야생형 수준으로 회복시켰다. ${\beta}TS$의 기질인 L-Ser는 다양한 효과를 나타낸다. 야생형과 NS104에서는 속도가 감소한 반면, GD51과 PH53에서는 거의 영향이 없었고, PT53와 DG56은 증가하였다. 이는 반응 중간 화학종의 분포의 변화와 연관될 가능성을 제시한다. GP와 L-Ser를 동시에 처리했을 때는 특이하게도 PH53는 가장 안정한 잔기치환체였다. 이는 Pro53가 소단위체간의 조절기작에서 중요한 역할을 하는 것을 시사한다.

Escherichia coli tryptophan synthase (TS) contains ${\alpha}_2{\beta}_2$, which catalyzes the final two steps in Trp biosynthesis. A molecular tunnel exists between the two active sites of ${\alpha}$ and ${\beta}$ subunits in TS. Via intersubunit communication, TS increases catalytic efficiency, including substrate channeling. The ${\beta}$ subunit of TS is composed of two domains, one of which, the COMM (communication) domain, plays an important role in intersubunit communication. The ${\alpha}$ subunit has a TIM barrel structure. This protein has functional regions at the C terminal of ${\beta}$ pleated sheets and in its loop regions. Three regions of the ${\alpha}$ subunit (${\alpha}L6$ [${\alpha}-loop$ L6], ${\alpha}L2$, and ${\alpha}L3$) are implicated in intersubunit communication. In the present study, conformational changes in ${\alpha}L6$ were monitored by measuring the sensitivity of mutant proteins in these regions to trypsin. The addition of a ${\alpha}$ subunit-specific ligand, D,L-${\alpha}$-glycerophosphate (GP), partially restored the sensitivity of mutant proteins to trypsin. In contrast, the addition of the ${\beta}$ subunit-specific ligand L-serine (Ser) resulted in varied sensitivity to trypsin, with an increase in PT53 (substitution of Pro with Thr at residue 53) and DG56, decrease in NS104 and wild type, and no change in GD51 and PH53. This finding may be related to several reaction intermediates formed under this condition. The addition of both GP and Ser led to a highly stable state of the complex. The present results are consistent with the current model. The method used herein may be useful for screening residues involved in intersubunit communication.

키워드

참고문헌

  1. Adachi, O., Kohn, L. D. and Miles, E. W. 1974. A rapid method for preparing crystalline ${\beta}$2 subunit of tryptophan synthase of Escherichia coli in high yield. J. Biol. Chem. 249, 7756-7763.
  2. Dunn, M. F. 2012. Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex. Arch. Biochem. Biophys. 519, 154-166. https://doi.org/10.1016/j.abb.2012.01.016
  3. Faeder, E. J. and Hammes, G. G. 1970. Kinetic studies of tryptophan synthase. Interaction of substrates with ${\beta}$ subunit. Biochemistry 9, 4043-4049. https://doi.org/10.1021/bi00823a003
  4. Higgins, W., Fairwell, T. and Miles, E. W. 1979. An active proteolytic derivative of the alpha subunit of tryptophan synthase: Identification of the site of cleavage and characterization of the fragment. Biochemistry 22, 4827-4835.
  5. Hilario, E., Caulkins, B. G., Huang, Y. M., You, W., Chang, C. A., Mueller, L. J., Dunn, M. F. and Fan, L. 2016. Visualizing the tunnel in tryptophan synthase with crystallography: Insights into a selective filter for accommodating indole and rejecting water. Biochim. Biophys. Acta 1864, 268-279. https://doi.org/10.1016/j.bbapap.2015.12.006
  6. Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W and Davies, D. R. 1988. Three-dimensional structure of the tryptophan synthase ${\alpha}$2${\beta}$2 multienzyme complex from Salmonella typhimurium. J. Biol. Chem. 267, 17857-17871.
  7. Jeong, M. S., Jeong, J. K., Park, K. S., Kim, H. T., Lee, K. M., Lim, W. K. and Jang, S. B. 2004. Crystallization and preliminary X-ray analysis of tryptophan synthase ${\alpha}$-subunits from Escherichia coli. Acta Crystallogr. D Biol. Crystallogr. 60, 132-134. https://doi.org/10.1107/S0907444903022935
  8. Kayastha, A. M., Sawa, U., Nagata S. and Miles, E. W. 1990. Site-directed mutagenesis of the ${\beta}$ subunit of tryptophan synthase from Salmonella typhimurium. J. Biol. Chem. 266, 7618-7825.
  9. Kim, J. W., Kim, E. Y., Park, H. H., Jung, J. E., Kim, H. D., Shin, H. J. and Lim, W. K. 2001. Homodimers of mutant tryptophan synthase ${\alpha}$-subunits in Escherichia coli. Biochem. Biophys. Res. Commun. 289, 568-572. https://doi.org/10.1006/bbrc.2001.6022
  10. Kirschner, K., Wiskocil, R. L., Foehn, M. and Rezeau, L. 1975. The tryptophan synthase from Escherichia coli. An improved purification procedure for the alpha-subunit and binding studies with substrate analogues. Eur. J. Biochem. 60, 513-523. https://doi.org/10.1111/j.1432-1033.1975.tb21030.x
  11. Miles, E. W., Yutani, K. and Ogasahara, K. 1982. Guanidine hydrochloride induced unfolding of the alpha subunit of tryptophan synthase and of the two alpha proteolytic fragments: evidence for stepwise unfolding of the two alpha domains. Biochemistry 21, 2586-2592. https://doi.org/10.1021/bi00540a002
  12. Leggett-Bailey, J. 1962. Techniques in Protein Chemistry. Elsevier Scientific Publishing Co. New York.
  13. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. https://doi.org/10.1038/227680a0
  14. Lim, W. K., Shin, H. J., Milton, D. L. and Hardman, J. K. 1991. Relative activities and stabilities of mutant Escherichia coli tryptophan synthase ${\alpha}$- subunits. J. Bacteriol. 173, 1886-1893. https://doi.org/10.1128/jb.173.6.1886-1893.1991
  15. Milton, D. L., Napier M. L., Muers, R. W. and Hardman, J. K. 1986. In vitro mutagenesis and overexpression of the Escherichia coli trpA gene and the partial characterization of the resultant tryptophan synthase mutant alpha subunits. J. Biol. Chem. 261, 16604-16615.