Acknowledgements: The authors would like to acknowledge Universiti Kebangsaan Malaysia for financial support through the GUP-2017-070, DIP-2012-13 and GGPM-2012-069 grants. This research is also supported by the Ministry of Higher Education, Malaysia through the ERGS/1/2011/STG/UKM/01/15 grant. We thank Diamond Light Source, UK for synchrotron beam time (I02 and I03) and we acknowledge Juan Sanchez-Weatherby and Thomas Sorensen for assistance at station I02 beamline during data collection. We thank Malaysia Genome and Vaccine Institute, National Institute of Biotechnology Malaysia for the in-house X-ray diffractometer facility. We thank Teo Chee How for useful discussion in phylogenetic analysis. We thank Pravin Kumran for technical support in protein purification. We also thank David W. Rice, Patrick J. Baker and Jon R. Sayers for insightful comments.Funder: Ministry of Higher Education, Malaysia for ERGS/1/2011/STG/UKM/01/15Burkholderia pseudomallei is a highly versatile pathogen with ~25% of its genome annotated to encode hypothetical proteins. One such hypothetical protein, BPSL1038, is conserved across seven bacterial genera and 654 Burkholderia spp. Here, we present a 1.55 Å resolution crystal structure of BPSL1038. The overall structure folded into a modified βαββαβα ferredoxin fold similar to known Cas2 nucleases. The Cas2 equivalent catalytic aspartate (D11) pairs are conserved in BPSL1038 although B. pseudomallei has no known CRISPR associated system. Functional analysis revealed that BPSL1038 is a nuclease with endonuclease activity towards double-stranded DNA. The DNase activity is divalent ion independent and optimum at pH 6. The concentration of monovalent ions (Na+ and K+) is crucial for nuclease activity. An active site with a unique D11(X20)SST motif was identified and proposed for BPSL1038 and its orthologs. Structure modelling indicates the catalytic role of the D11(X20)SST motif and that the arginine residues R10 and R30 may interact with the nucleic acid backbone. The structural similarity of BPSL1038 to Cas2 proteins suggests that BPSL1038 may represent a sub-family of nucleases that share a common ancestor with Cas2
