Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Conservative Tryptophan Residue in the Vicinity of an Active Site of the M15 Family L, D-peptidases: A Key Element in the Catalysis

Galina V. Mikoulinskaia
* ORCID logo ,
Dmitry A. Prokhorov
,
Sergei V. Chernyshov
,
Daria S. Sitnikova
,
Arina G. Arakelian
,
Vladimir N. Uversky
* ORCID logo
Version 1 : Received: 27 July 2023 / Approved: 28 July 2023 / Online: 28 July 2023 (12:29:31 CEST)

A peer-reviewed article of this Preprint also exists.

Mikoulinskaia, G.V.; Prokhorov, D.A.; Chernyshov, S.V.; Sitnikova, D.S.; Arakelian, A.G.; Uversky, V.N. Conservative Tryptophan Residue in the Vicinity of an Active Site of the M15 Family l,d-Peptidases: A Key Element in the Catalysis. Int. J. Mol. Sci. 2023, 24, 13249. Mikoulinskaia, G.V.; Prokhorov, D.A.; Chernyshov, S.V.; Sitnikova, D.S.; Arakelian, A.G.; Uversky, V.N. Conservative Tryptophan Residue in the Vicinity of an Active Site of the M15 Family l,d-Peptidases: A Key Element in the Catalysis. Int. J. Mol. Sci. 2023, 24, 13249.

Abstract

Bioinformatics analysis of the sequences of orthologous zinc-containing peptidases of the M15_C subfamily revealed the presence of a conserved tryptophan residue near the active site, which is not involved in the formation of the protein core. Site-directed mutagenesis of this Trp114/109 residue using two representatives of the family, L-alanoyl-D-glutamate peptidases of bacteriophages T5 (Calcium-activated EndoT5) and RB49 (EndoRB49, without ion regulation) as an example, and further analysis of the 1H NMR spectra of the mutants showed that a decrease in the volume of the W → F → A residue leads to changes in the hydrophobic core and active center of the protein, and also decreased affinity for regulatory Ca2+ in the EndoT5 mutants. The inactive T5W114A mutant lacks the ability to bind the substrate. In general, the conserved Trp114/109 residue, due to the spatial restrictions of its side chain, significantly affects the formation of the catalytically active form of the enzyme and is critical for catalysis.

Keywords

L,D-peptidase; bacteriophage; catalysis; substrate binding; calcium

Subject

Biology and Life Sciences, Biochemistry and Molecular Biology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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