@misc{10481/106601,
year = {2025},
month = {8},
url = {https://hdl.handle.net/10481/106601},
abstract = {M2-32 is a non-specific acid phosphatase with a rare ability to function
across a broad pH range (3.5–8.5). Analysis using SWISS-PROT Prf Profiles classifies it as a class A acid phosphatase (Z-score: 78.97), sharing
50%–60% sequence similarity with enzymes such as PhoC and PhoN. For
detailed characterization, the gene encoding M2-32 was cloned into the
pET28(b) vector, overexpressed in Escherichia coli BL21 (DE3), and subsequently purified. Although the monomeric form of M2-32 has a molecular
weight of  28 kDa, size exclusion chromatography, dynamic light scattering, and sedimentation studies revealed a dimeric form in solution. Enzymatic assays using p-nitrophenyl phosphate, 4-methylumbelliferyl
phosphate, 30
-and 50
-adenosine monophosphate demonstrated robust activity over a pH range of 4.0–8.0 at both 30 and 50C. Differential scanning
fluorimetry indicated an unfolding temperature close to 47C; however, the
enzyme refolded after heat denaturation at 80C. We have determined the
x-ray crystal structure of M2-32 by molecular replacement using an
AlphaFold2-guided truncated model, achieving a resolution of 2.2 Å. The
protein crystallized as a dimer-of-dimers. Each monomer (residues 38–274)
adopts an all-alpha-helical fold composed of 14 helices and two disulfide
bonds. Docking studies with adenosine monophosphates, combined with
site-directed mutagenesis, identified His174, Arg207, His213, Asp217 as
critical catalytic residues, and Tyr136 and Ser172 probably involved in substrate recognition. Mutations at these positions resulted in over 90% loss of
enzymatic activity, highlighting their functional significance.},
organization = {Agencia Estatal de Investigacion (Grant/Award Number: PDI-2021-123469OBI00)},
organization = {Gobierno Regional de Andalucía (Grant/Award Number: P20-00049)},
publisher = {Wiley Periodicals LLC},
keywords = {Bacteria},
keywords = {bacterial acid phosphatase},
keywords = {biomineralization},
title = {Thermotolerant class A acid phosphatase active across broad pH range and diverse substrates},
doi = {10.1002/pro.70244},
author = {Recio Muñoz, María Isabel and Gavira Gallardo, José Antonio and de la Torre, Jesús and Cano-Muñoz, Mario and Martínez Rodríguez, Sergio and Daddaoua, Abdelali and Duque, Estrella and Ramos, Juan Luis},
}