Abstract
Genomic sequence analysis of Acinetobacter baumannii revealed the presence of a putative Acid Phosphatase (AcpA; EC 3.1.3.2). A plasmid construct was made, and recombinant protein (rAcpA) was expressed in E. coli. PAGE analysis (carried out under denaturing/reducing conditions) of nickel-affinity purified protein revealed the presence of a near-homogeneous band of approximately 37 kDa. The identity of the 37 kDa species was verified as rAcpA by proteomic analysis with a molecular mass of 34.6 kDa from the deduced sequence. The dependence of substrate hydrolysis on pH was broad with an optimum observed at 6.0. Kinetic analysis revealed relatively high affinity for PNPP (Km = 90 μM) with Vmax, kcat, and Kcat/Km values of 19.2 pmoles s-1, 4.80 s-1(calculated on the basis of 37 kDa), and 5.30 x 104 M-1s-1, respectively. Sensitivity to a variety of reagents, i.e., detergents, reducing, and chelating agents as well as classic acid phosphatase inhibitors was examined in addition to assessment of hydrolysis of a number of phosphorylated compounds. Removal of phosphate from different phosphorylated compounds is supportive of broad, i.e., ‘nonspecific’ substrate specificity; although, the enzyme appears to prefer phosphotyrosine and/or peptides containing phosphotyrosine in comparison to serine and threonine. Examination of the primary sequence indicated the absence of signature sequences characteristic of Type A, B, and C nonspecific bacterial acid phosphatases.
Highlights
Phosphatases (EC 3.1.3) are a diverse, ubiquitous group of enzymes that hydrolyze phosphoesters from a wide variety of compounds [1]
C-terminus His(6x)-tagged Acinetobacter baumannii recombinant acid phosphatase (rAcpA) migrated as a monomer of ~37 kDa under reducing denaturing conditions which is in good agreement with the predicted molecular weight from protein sequence (Fig 1B), i.e., 34,619
The bipartite Class C signature sequence with defining aspartic acid residues is missing in Acinetobacter baumannii rAcpA, the primary sequence does contain multiple aspartic acid (D) residues
Summary
Phosphatases (EC 3.1.3) are a diverse, ubiquitous group of enzymes that hydrolyze phosphoesters from a wide variety of compounds [1]. These enzymes have been broadly grouped according to their pH optimum, i.e., acid, neutral or alkaline. Acid phosphatases have been further grouped according to molecular weight, substrate specificity, response to inhibitors, physical appearance, and sequence homology into two large families, i.e., specific and nonspecific. Both are widely distributed among Gram-positive and Gram-negative bacteria.
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