Abstract
Cyclodipeptide synthases (CDPSs) form various cyclodipeptides from two aminoacyl tRNAs via a stepwise mechanism with the formation of a dipeptidyl enzyme intermediate. As a final step of the catalytic reaction, the dipeptidyl group undergoes intramolecular cyclization to generate the target cyclodipeptide product. In this work, we investigated the cyclization reaction in the cyclodipeptide synthase AlbC using QM/MM methods and free energy simulations. The results indicate that the catalytic Y202 residue is in its neutral protonated form, and thus, is not likely to serve as a general base during the reaction. We further demonstrate that the reaction relies on the conserved residue Y202 serving as a proton relay, and the direct proton transfer from the amino group to S37 of AlbC is unlikely. Calculations reveal that the hydroxyl group of tyrosine is more suitable for the proton transfer than hydroxyl groups of other amino acids, such as serine and threonine. Results also show that the residues E182, N40, Y178 and H203 maintain the correct conformation of the dipeptide needed for the cyclization reaction. The mechanism discovered in this work relies on the amino groups conserved among the entire CDPS family and, thus is expected to be universal among CDPSs.
Highlights
Cyclodipeptide synthases (CDPSs) are a family of enzymes that use aminoacyl-tRNAs to synthetize cyclodipeptides, which are precursors of many secondary metabolites with diverse biological functions[1,2]
The catalytic reaction starts with the binding of the first aa-tRNA and the transfer of its aminoacyl moiety to a conserved serine residue leading to the formation of an aminoacyl enzyme intermediate
It was demonstrated that the Y202F mutation does not affect the formation of the aminoacyl enzyme intermediate[5], but leads to the accumulation of the covalent dipeptidyl enzyme intermediate, which was not observed with the wild-type enzyme[7]
Summary
The computed free energy associated with the proton transfer is 2.7 ± 0.8 kcal/mol favoring the state with the neutral amino group and neutral tyrosine. To test the effect of the enzyme environment on the stabilization of the neutral amino group we recomputed the free energy of the proton transfer in vacuum and in the COSMO implicit solvent[8] using the geometry of the QM region taken from the protein simulations. The computed energy for the proton transfer from the tyrosine to the amino group is 14.5 kcal/mol and 7.1 kcal/mol in vacuum and in the implicit solvent, respectively, both favoring the state with the neutral tyrosine. The state with the charged tyrosine form is not favored in the protein and in the solvent, but the enzyme environment strongly favors the proton transfer from Y202 to the amino group by 4.4 kcal/mol relative to solvent
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
