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 . 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 , 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  and the structure model was refined in REFMAC5 .
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.
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).