ApaH diadenosine polyphosphatase from Legionella pneumophila

Abstract

The Nudix diadenosine polyphosphatase, NudA, is a virulence factor in Legionella pneumophila (LP), the causative agent of Legionnaires’ disease. The nonNudix diadenosine polyphosphatase, ApaH, is a virulence factor in E. coli and acts additively with the E. coli NudA for virulence. An LP apaH knockout mutant had no apparent phenotype; however, a double apaH/nudA knockout mutant could not be created, indicating that this dual mutation is lethal, and that apaH is important. We subcloned apaH, but ApaH was insoluble, so we subcloned apaH using the maltose binding protein (MBP) as a solubility tag. We obtained a soluble fusion protein with diadenosine polyphosphatase activity. Neither a MBP nor His tag could be used for purification indicating that the protein may not be folded properly, and thus we are in the process of subcloning the apaH, once again, to put the MBP solubility tag at the c‐terminal end instead of the n‐terminal end where it was originally placed. Once the ApaH diadenosine polyphosphatase has been purified, it will be characterized further enzymatically. This research has been supported by an NIH AREA grant and the Merck/AAAS Undergraduate Science Research Program.