A histidine acid phosphatase (HAP) (PhySc) with 99.50% protein sequence similarity with PHO5 from Saccharomyces cerevisiae was expressed functionally with the molecular mass of approximately 110 kDa through co-expression along with the set of molecular chaperones dnaK, dnaJ, GroESL. The purified HAP illustrated the optimum activity of 28.75 +/- 0.39 U/mg at pH 5.5 and 40 C. The K(m) and K(cat) values towards calcium phytate were 0.608 +/- 0.09 mM and 650.89 +/- 3.6 s(- 1). The half-lives (T(1/2)) at 55 and 60 C were 2.75 min and 55 s, respectively. The circular dichroism (CD) demonstrated that PhySc includes 30.5, 28.1, 21.3, and 20.1% of random coils, alpha-Helix, beta-Turns, and beta-Sheet, respectively. The T(m) recorded by CD for PhySc was 56.5 +/- 0.34 C. The molecular docking illustrated that His59 and Asp322 act as catalytic residues in the PhySc. MD simulation showed that PhySc at 40 C has higher structural stability over those of the temperatures 60 and 80 C that support the thermodynamic in vitro investigations. Secondary structure content results obtained from MD simulation indicated that PhySc consists of 34.03, 33.09, 17.5, 12.31, and 3.05% of coil, helix, turn, sheet, and helix310, respectively, which is almost consistent with the experimental results.

• PMID: 38630327
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Journal Article

Authors

Nezhad NG, Jamaludin SZB, Rahman RNZRA, Yahaya NM, Oslan SN, Shariff FM, Isa NM, Leow TC

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