TY - JOUR
T1 - Biochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii
AU - Smiley-Moreno, Elizabeth
AU - Smith, Douglas
AU - Yu, Jieh Juen
AU - Cao, Phuong
AU - Arulanandam, Bernard P.
AU - Chambers, James P.
N1 - Funding Information:
This study was supported by UTSA Grant G02312 (JPC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Publisher Copyright:
Copyright © 2021 Smiley-Moreno et al.
PY - 2021/6
Y1 - 2021/6
N2 - 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 nearhomogeneous 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 × 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.
AB - 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 nearhomogeneous 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 × 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.
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U2 - 10.1371/journal.pone.0252377
DO - 10.1371/journal.pone.0252377
M3 - Article
C2 - 34077475
AN - SCOPUS:85107318236
VL - 16
JO - PLoS One
JF - PLoS One
SN - 1932-6203
IS - 6 June
M1 - e0252377
ER -