1. A Genetically Encoded Two-Dimensional Infrared Probe for Enzyme Active-Site Dynamics
Li Wang, et al. Angew Chem Int Ed Engl. 2021 May 10;60(20):11143-11147. doi: 10.1002/anie.202016880. Epub 2021 Apr 7.
While two-dimensional infrared (2D-IR) spectroscopy is uniquely suitable for monitoring femtosecond (fs) to picosecond (ps) water dynamics around static protein structures, its utility for probing enzyme active-site dynamics is limited due to the lack of site-specific 2D-IR probes. We demonstrate the genetic incorporation of a novel 2D-IR probe, m-azido-L-tyrosine (N3Y) in the active-site of DddK, an iron-dependent enzyme that catalyzes the conversion of dimethylsulfoniopropionate to dimethylsulphide. Our results show that both the oxidation of active-site iron to FeIII , and the addition of denaturation reagents, result in significant decrease in enzyme activity and active-site water motion confinement. As tyrosine residues play important roles, including as general acids and bases, and electron transfer agents in many key enzymes, the genetically encoded 2D-IR probe N3Y should be broadly applicable to investigate how the enzyme active-site motions at the fs-ps time scale direct reaction pathways to facilitating specific chemical reactions.
2. 3-azido-L-tyrosine as a photoinhibitor of tubulin:tyrosine ligase. Role of thiol groups
K Coudijzer, M Joniau FEBS Lett. 1990 Jul 30;268(1):95-8. doi: 10.1016/0014-5793(90)80981-n.
We have synthesized the photoactivatable probes 3-azido-L-tyrosine and p-azido-L-phenylalanine and studied their capacity to inhibit the incorporation of [3H]tyrosine into tubulin catalyzed by tubulin:tyrosine ligase. Without illumination, only 3-azido-L-tyrosine reversibly inhibits the enzyme. Upon illumination, both reagents irreversibly photoinactivate the enzyme in a similar way. The ligase can be protected against photoinactivation by reversibly blocking essential thiol groups with pCMB during illumination.
3. Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion
Fumie Iraha, Kenji Oki, Takatsugu Kobayashi, Satoshi Ohno, Takashi Yokogawa, Kazuya Nishikawa, Shigeyuki Yokoyama, Kensaku Sakamoto Nucleic Acids Res. 2010 Jun;38(11):3682-91. doi: 10.1093/nar/gkq080. Epub 2010 Feb 16.
Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase-tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)-tRNA(Tyr) pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS-tRNA(Tyr) pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNA(Tyr). The endogenous TyrRS and tRNA(Tyr) genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS-tRNA(Tyr) pair. In this engineered strain, 3-iodo-L-tyrosine and 3-azido-L-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-L-tyrosine and was also found to recognize 3-azido-L-tyrosine. The structural basis for the 3-azido-L-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.
