Expression of a Recombinant Acid Phosphatase from Acinetobacter baumannii

Phosphatases constitute a diverse, ubiquitous group of enzymes that hydrolyze various phosphoester substrates, and are widely distributed among Gram positive and negative bacteria. Acinetobacter baumannii is an opportunistic pathogen becoming a medically relevant nosocomial pathogen accounting for approximately 2 % of all healthcare associated infections in the United States. Most importantly, the increase in multidrug resistant Acinetobacter phenotypes has prompted the Centers for Disease Control to list Acinetobacter as a 'severe’ threat requiring use of last line treatment options.

Genomic sequence data suggest the presence of a putative Acinetobacter Acid Phosphatase (NCBI Reference Sequence WP_000749225.1). Because Acid Phosphatases (EC 3.1.3.2) have been implicated as virulence factors, and given the emergence of this pathogen, we were interested in cloning this enzyme from Acinetobacter baumannii. Utilizing the pET expression system, a poly His-tag gene construct was expressed in E. coli, and recombinant protein purified on Ni-NTA Agarose beads. Under denaturing conditions, SDS-PAGE analysis revealed the 250 mM imidazole Ni-NTA Agarose bead eluate to migrate as a homogenous band of approximately 35 kDa. Mass spectroscopic sequence analysis confirmed the recombinant protein’s identity as that of a bacterial acid phosphatase. Zymogram PAGE analysis revealed the catalytic species capable of hydrolyzing 4-methylumbelliferyl phosphate to be of approximately 80 kDa in size. Hydrolysis of 4-methylumbelliferyl phosphate carried out at pH 6.5 (200 mM Acetate buffer) is optimal in the presence of either 2.0 mM Co++ or Ni++ chloride. Chlorides of Hg++ and Cu++ were observed to be very inhibitory. Kinetic analysis of 4-methylumbelliferyl phosphate hydrolysis revealed Km and Vmax values of 164 μM and 5,800 nmoles/minute, respectively. Although the Acid Phosphatase of Acinetobacter baumannii exhibits high affinity for the artificial substrate 4-methylumbelliferyl phosphate, the enzyme’s native endogenous substrate, remains unknown.