Tyrosine and Pyrosyl-Lysine tRNA/aminoacyl-synthetase
Tyrosine tRNA/aminoacyl-synthetase
There are several amino acids which have been incorporated into peptides and proteins in Escherichia coli . Among others, it was possible to incorporate ncAA with heavy atoms, keto and alkaline side chains, photo crosslinking and so on (Zhang et al.2005).
For the binding of the ncAA, an adaption of the synthetase is necessary. This is done by certain mutations of several regions of the synthetase, leading to a conformational change of the binding pocket and resulting in the binding of a new amino acid.
The tyrosine synthetase is the first orthogonal E. coli tRNA/aaRS pair generated from archaea and the best known so far (Wang et al.2001). This tyrosine synthetase (TyrRS) has a small anticodon loop binding domain (Steer et al.1999), so the change of the recognition in order to change the anticodon loop of its cognate tRNA to CUA is not as difficult as with a larger anticodon loop binding domain (Steer et al.1999). The binding side is located deep inside a small pocket of the TyrRS (Tian et al.2004). This leads to assume, that no large conformational changes are necessary for the incorporation of the ncAA. Rather very small conformational changes result in a larger or smaller binding pocket and an altered hydrogen-binding interaction with the ligand (Zhang et al.2005).
The TyrRS binding pocket is highly hydrophilic, preventing the binding of phenylalanine, whose structure is similar to the one of tyrosine (Goldgur et al.1997). Changing the characteristic of the binding pocket into a hydrophobic kind, could favor the binding of phenylalanine derivates (Goldgur et al.1997). In the middle of the negative charged binding pocket (Zhang et al.2005), there is an aspartic acid on position 158. The entrance of the binding pocket is positioned between two glutamic acids at positions 36 and 172. Upon tRNA binding, the aspartic amino acid Asp158 forms two hydrogen bonds with the glycine at position 34 (Zhang et al.2005).
When the ligand tyrosine is bound, hydrogen bonds to the Tyr32, Asp158, Glu36, Gln173, Tyr151 and Gln155 are formed, resulting in subtle movements of the side chains within the tyrosine-binding pocket (Zhang et al.2005). Beside the binding pocket, also other domains are affected by the binding of the tyrosine. For example, the loop 73-83, positioned at the entrance of the binding pocket, which due to the conformational changes then provides a hydrophobic lid over the binding pocket. This is assumed to have the effect of separating the activated tyrosine from water during the catalytic reaction (Zhang et al.2005).
Pyrosyl-Lysine tRNA/aminoacyl-synthetase
