Site-directed mutagenesis of varicella-zoster virus thymidylate synthase. Analysis of two highly conserved regions of the enzyme.
作者： Patrick T. Harrison , James E. Scott , Michael J. Hutchinson , Russell Thompson
DOI: 10.1111/J.1432-1033.1995.0511H.X
关键词: Thymidylate synthase 、 Site-directed mutagenesis 、 Glycine 、 Amino acid 、 Mutagenesis (molecular biology technique) 、 Biology 、 Active site 、 Residue (chemistry) 、 Molecular biology 、 Enzyme 、 Biochemistry
摘要: We have constructed a series of mutants to study the role two structurally and functionally important regions thymidylate synthase (TS) from varicella-zoster virus (VZV). The first centres on conserved glycine residue in β-kink β-strand i, partially buried region protein that is for dimer interactions formation active site. show located i not essential enzyme activity can readily accommodate several amino acid substitutions also an insertion. A covariant accommodates these changes was identified. second interest solvent-exposed highly mobile C-terminal which component site TS Lactobacillus casei Escherichia coli. demonstrate removal VZV does completely inactivate enzyme, implying there are significant structural differences between bacterial enzymes. By combining site-directed mutagenesis molecular modelling we identified propose model explains contrasting activities.
sci-hub.se 参考文章(35)
D. Gonzalez-Pacanowska, S. Climie, R. Stroud, D. V. Santi, Sung-Woo Cho, L. Ruiz-Perez, P. Prapunwattana, Saturation site-directed mutagenesis of thymidylate synthase. Journal of Biological Chemistry. ,vol. 265, pp. 18776- 18779 ,(1990) , 10.1016/S0021-9258(17)30579-3
F. William Studier, Alan H. Rosenberg, John J. Dunn, John W. Dubendorff, Use of T7 RNA polymerase to direct expression of cloned genes. Methods in Enzymology. ,vol. 185, pp. 60- 89 ,(1990) , 10.1016/0076-6879(90)85008-C
Robert M. Stroud, Janet S. Finer‐Moore, Stereochemistry of a multistep/bipartite methyl transfer reaction: thymidylate synthase. The FASEB Journal. ,vol. 7, pp. 671- 677 ,(1993) , 10.1096/FASEBJ.7.8.8500692
L. Hardy, J. Finer-Moore, W. Montfort, M. Jones, D. Santi, R. Stroud, Atomic structure of thymidylate synthase: target for rational drug design Science. ,vol. 235, pp. 448- 455 ,(1987) , 10.1126/SCIENCE.3099389
A. T. BRUNGER, J. KURIYAN, M. KARPLUS, Crystallographic R Factor Refinement by Molecular Dynamics Science. ,vol. 235, pp. 458- 460 ,(1987) , 10.1126/SCIENCE.235.4787.458
C. T. Chung, S. L. Niemela, R. H. Miller, One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proceedings of the National Academy of Sciences of the United States of America. ,vol. 86, pp. 2172- 2175 ,(1989) , 10.1073/PNAS.86.7.2172
D.A. Matthews, K. Appelt, S.J. Oatley, Stacked beta-bulges in thymidylate synthase account for a novel right-handed rotation between opposing beta-sheets. Journal of Molecular Biology. ,vol. 205, pp. 449- 454 ,(1989) , 10.1016/0022-2836(89)90354-9
Marjorie Russel, Simon Kidd, Mark R. Kelley, An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene. ,vol. 45, pp. 333- 338 ,(1986) , 10.1016/0378-1119(86)90032-6
Shane C. Climie, Christopher W. Carreras, Daniel V. Santi, Complete replacement set of amino acids at the C-terminus of thymidylate synthase: quantitative structure-activity relationship of mutants of an enzyme. Biochemistry. ,vol. 31, pp. 6032- 6038 ,(1992) , 10.1021/BI00141A011
I. D. Kuntz, Structure-based strategies for drug design and discovery. Science. ,vol. 257, pp. 1078- 1082 ,(1992) , 10.1126/SCIENCE.257.5073.1078