Biochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii.

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 pu...

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Autores principales:	Elizabeth Smiley-Moreno, Douglas Smith, Jieh-Juen Yu, Phuong Cao, Bernard P Arulanandam, James P Chambers
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a5cfc8dee3664237a25988adca4bcd40
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spelling	oai:doaj.org-article:a5cfc8dee3664237a25988adca4bcd402021-12-02T20:05:21ZBiochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii.1932-620310.1371/journal.pone.0252377https://doaj.org/article/a5cfc8dee3664237a25988adca4bcd402021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0252377https://doaj.org/toc/1932-6203Genomic 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.Elizabeth Smiley-MorenoDouglas SmithJieh-Juen YuPhuong CaoBernard P ArulanandamJames P ChambersPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 6, p e0252377 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Elizabeth Smiley-Moreno Douglas Smith Jieh-Juen Yu Phuong Cao Bernard P Arulanandam James P Chambers Biochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii.
description	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.
format	article
author	Elizabeth Smiley-Moreno Douglas Smith Jieh-Juen Yu Phuong Cao Bernard P Arulanandam James P Chambers
author_facet	Elizabeth Smiley-Moreno Douglas Smith Jieh-Juen Yu Phuong Cao Bernard P Arulanandam James P Chambers
author_sort	Elizabeth Smiley-Moreno
title	Biochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii.
title_short	Biochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii.
title_full	Biochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii.
title_fullStr	Biochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii.
title_full_unstemmed	Biochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii.
title_sort	biochemical characterization of a recombinant acid phosphatase from acinetobacter baumannii.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/a5cfc8dee3664237a25988adca4bcd40
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Biochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii.

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