MS09-P13 Bacterial metal-dependent DNase with novel fold Jan Dohnalek (Laboratory of Structure and function of biomolecules, Institute of Biotechnology of the Czech Academy of Sciences, Vestec, Czech Republic) Jan Stránský (Laboratory of Structure and function of biomolecules, Institute of Biotechnology of the Czech Academy of Sciences, Vestec, Czech Republic) Vojtěch Andrle (Laboratory of Structure and function of biomolecules, Institute of Biotechnology of the Czech Academy of Sciences, Vestec, Czech Republic) Mária Trundová (Laboratory of Structure and function of biomolecules, Institute of Biotechnology of the Czech Academy of Sciences, Vestec, Czech Republic) Tomáš Kovaľ (Laboratory of Structure and function of biomolecules, Institute of Biotechnology of the Czech Academy of Sciences, Vestec, Czech Republic) Lars Østergaard (Department of Agile Protein Screening, Novozymes A/S, Bagsvaerd, Denmark)email: dohnalek007@gmail.comBacillus licheniformis produces extracellular DNase NucB capable of double-strand DNA cleavage [1]. NucB is a sporulation-specific enzyme with so far unknown specificity and structure-function properties. NucB was successfully produced using bacterial expression, purified, crystallized and diffraction data were collected using synchrotron radiation and a MetalJet in-house X-ray source [2]. Systematic optimization of the key S-SAD protocol parameters was necessary to solve the phase problem [2]. The enzyme structure determined in several crystal forms uncovers a new nuclease/DNase fold. Structural analysis, bioinformatic analysis, and activity measurements for the wt and mutant forms of the enzyme led to conclusions regarding the position and composition of the active site. Detailed metal-dependence, stability and cleavage specificity profiles show the necessity of divalent ions for the activity, high temperature stability, and a time-dependent preference for the formation of single strand breaks on dsDNA substrate at sequence-specific sites. References:

[1] van Sinderen, D., Kiewiet, R., Venema, G. (1995). Differential Expression of 2 Closely-related Deoxyribonuclease Genes, NucA and NucB, in Bacillus subtilis. Molecular Microbiology, 15, 213–223.

[2] Stransky, J., Optimization of Macromolecular Crystallography Tools and Structural Studies of Nucleases, PhD dissertation, Czech Technical University in Prague, 2018.

Keywords: DNase, metal-dependent, X-ray structure