Integrative structural analysis of antibiotic-inactivating enzyme from Stenotrophomonas maltophilia

M. Malý1,2, P. Kolenko1,2, J. Dušková1, T. Kovaľ1, T. Skálová1, M. Trundová1, J. Stránský1, L. Švecová1, K. Adámková1,3, B. Husťáková1,3, J. Dohnálek1

1Institute of Biotechnology of the Czech Academy of Sciences, Biocev, Průmyslová 595, Vestec

2Czech Technical University in Prague, Faculty of Nuclear Sciences and Physical Engineering, Břehová 7, Prague

3University of Chemical and Technology Prague, Department of Biochemistry and Microbiology, Technická 5, Prague

Stenotrophomonas maltophilia is an emerging pathogenic bacterium that causes an increasing number of serious infections worldwide. Mutations and the acquisition of antibiotic-resistance genes were shown to extend the broad antibiotic resistance of this species [1]. We performed a bioinformatic analysis of its available genomes to discover uncharacterised antibiotic-inactivating enzymes to carry out functional and structural analyses.

The chosen target enzymes were expressed in Escherichia coli and successfully purified. To confirm the expected function – enzymatic inactivation of antibiotics, kinetic assays were performed. An enzyme catalysing the antibiotic-inactivation reaction was crystallized and diffraction images were collected. The dataset exhibits severe anisotropy: an estimated resolution limit in Aimless [2], according to the criterion to I/σ(I) > 1.5, varied from 2.69 Å to 1.96 Å for different directions in reciprocal lattice. The phase problem was solved with MoRDa [3] and the structure model was refined in REFMAC5 [4].

Surprisingly, the crystal structure consists of a homodimer covalently linked via two cysteine bridges. However, consequent integrative structural analysis using SAXS, MS, MX and DLS indicates a monomeric state in solution. Nevertheless, the determined atomic structure reveals a spatial arrangement of the active site in detail. This provides an important knowledge for the development of antibiotic treatment strategies, as well as for further structural analysis – in vitro or in silico – of complexes with antibiotics or potential enzyme inhibitors.

1. T. Gil-Gil, J. L. Martínez, P. Blanco, Expert Review of Anti-infective Therapy, 18, (2020), pp. 335-347.

2. P .R. Evans, G. N. Murshudov, Acta Cryst., D69, (2013), pp. 1204-1214.

3. A. Vagin, A. Lebedev, Acta Cryst., A71, (2015), s19.

4. G. N. Murshudov, P. Skubak, A. A. Lebedev, N. S. Pannu, R. A. Steiner, R. A. Nicholls, M. D. Winn, F. Long, A. A. Vagin, Acta Cryst., D67, (2011), pp. 355-367.

This work was supported by the MEYS CR (projects CAAS – CZ.02.1.01/0.0/0.0/16_019/0000778, BIOCEV – CZ.1.05/1.1.00/02.0109, and ELIBIO – CZ.02.1.01/0.0/0.0/15_003/0000447) from the ERDF fund; by the Czech Academy of Sciences (86652036); by the GA CTU in Prague (SGS22/114/OHK4/2T/14); by the Czech Science Foundation (20-12109S); and from the grant of Specific university research (A1_FPBT_2021_003). We acknowledge CMS-Biocev (Biophysical techniques, Crystallization, Diffraction, Structural mass spectrometry) supported by MEYS CR (LM2015043 and LM2018127).