Comparative analysis of two non-specific nucleases of the phospholipase D family from the plant pathogen competitor bacterium Pantoea agglomerans

Abstract

Bacterial non-specific nucleases of the phospholipase D family are widely distributed among the members of the Enterobacteriaceae. Each genome mainly contains a single copy of a gene encoding a phospholipase D family protein. However, two distantly related isozymes (< 40% identity at the protein level) were identified by BLAST-analyses in the plant pathogenic competitor enterobacterium Pantoea agglomerans. The two nucleases PaNuc-1 and PaNuc-2 were produced in Escherichia coli. Identical gene constructs and expression conditions resulted in the production of PaNuc-1 in soluble form, while PaNuc-2 remained insoluble in inclusion bodies. PaNuc-2 was refolded and both proteins were purified by a combination of affinity and ion exchange chromatography. Proteolytic removal of the HIS-tag allowed the characterization of pure and mature tag-less proteins. Enzymatic properties of both isozymes revealed that they are non-specific nucleases, displaying activities against RNA, single- and double-stranded genomic DNA as well as circular plasmids. However, their biochemical activity profiles were clearly different, with PaNuc-1 being optimally active at 70 °C and pH 7.0, while PaNuc-2 was most active at 45 °C and pH 7.0. The enzymes retained > 90% nuclease activity at EDTA concentrations of 4 mM (PaNuc-2) and 20 mM (PaNuc-1), respectively. Different enzymatic properties suggest that the roles of PaNuc-1 and PaNuc-2 differ in the cell and might be the result of functional diversification after an ancient gene duplication event took place. The fact that both enzymes could be easily produced in recombinant form and their tolerance against metal ion chelators in combination with a broad substrate promiscuity might pave the way to versatile biotechnological applications.

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