Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Sep 26;23(19):11319.
doi: 10.3390/ijms231911319.

Improving Both the Thermostability and Catalytic Efficiency of Phospholipase D from Moritella sp. JT01 through Disulfide Bond Engineering Strategy

Lilang Li  1 , Xuejing Mao  1 , Fuli Deng  1 , Yonghua Wang  1 , Fanghua Wang  1
Affiliations

Improving Both the Thermostability and Catalytic Efficiency of Phospholipase D from Moritella sp. JT01 through Disulfide Bond Engineering Strategy

Lilang Li et al. Int J Mol Sci. .

Abstract

Mining of Phospholipase D (PLD) with high activity and stability has attracted strong interest for investigation. A novel PLD from marine Moritella sp. JT01 (MsPLD) was biochemically and structurally characterized in our previous study; however, the short half-life time (t1/2) under its optimum reaction temperature seriously hampered its further applications. Herein, the disulfide bond engineering strategy was applied to improve its thermostability. Compared with wild-type MsPLD, mutant S148C-T206C/D225C-A328C with the addition of two disulfide bonds exhibited a 3.1-fold t1/2 at 35 °C and a 5.7 °C increase in melting temperature (Tm). Unexpectedly, its specific activity and catalytic efficiency (kcat/Km) also increased by 22.7% and 36.5%, respectively. The enhanced activity might be attributed to an increase in the activation entropy by displacing more water molecules by the transition state. The results of molecular dynamics simulations (MD) revealed that the introduction of double disulfide bonds rigidified the global structure of the mutant, which might cause the enhanced thermostability. Finally, the synthesis capacity of the mutant to synthesize phosphatidic acid (PA) was evaluated. The conversion rate of PA reached about 80% after 6 h reaction with wild-type MsPLD but reached 78% after 2 h with mutant S148C-T206C/D225C-A328C, which significantly reduced the time needed for the reaction to reach equilibrium. The present results pave the way for further application of MsPLD in the food and pharmaceutical industries.

Keywords: disulfide bonds; molecular dynamics simulations; phosphatidic acid; phospholipase D; protein engineering; thermostability.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Thermoactivity of wild-type MsPLD and various mutants. Effect of temperature on the hydrolysis activity of wild-type MsPLD and mutants S148C-T206C, D225C-A328C and S148C-T206C/D225C-A328C.
Figure 2
Figure 2
Three-dimensional model of the mutant S148C-T206C/D225C-A328C. The structural model of mutant S148C-T206C/D225C-A328C was built with Modeller, and the crystal structure of wild-type MsPLD (PDB ID 7WU1) was used as the template. Introduced disulfide bonds S148C-T206C and D225C-A328C were shown as orange sticks. The catalytic residues H258 and H498 were shown as magenta sticks.
Figure 3
Figure 3
MD simulation analysis of wild-type MsPLD and mutant S148C-T206C/D225C-A328C. (A) Time courses of the fraction of native contacts (Q). (B) The difference in Cα RMSF of mutant S148C-T206C/D225C-A328C versus that of wild-type MsPLD. (C) Time courses of RMSD value. (D) The relative frequency distributions of Rg. (E) The relative frequency distributions of hydrophobic SASA.
Figure 4
Figure 4
Time course of the production of PA from PC by wild-type MsPLD and mutant S148C-T206C/D225C-A328C.
See this image and copyright information in PMC

References

    1. Xu P., Ni Z.F., Zong M.H., Ou X.Y., Yang J.G., Lou W.Y. Improving the thermostability and activity of Paenibacillus pasadenensis chitinase through semi-rational design. Int. J. Biol. Macromol. 2020;150:9–15. doi: 10.1016/j.ijbiomac.2020.02.033. - DOI - PubMed
    1. Tizei P.A., Csibra E., Torres L., Pinheiro V.B. Selection platforms for directed evolution in synthetic biology. Biochem. Soc. Trans. 2016;44:1165–1175. doi: 10.1042/BST20160076. - DOI - PMC - PubMed
    1. Zhang H., Sang J., Zhang Y., Sun T., Liu H., Yue R., Zhang J., Wang H., Dai Y., Lu F., et al. Rational design of a Yarrowia lipolytica derived lipase for improved thermostability. Int. J. Biol. Macromol. 2019;137:1190–1198. doi: 10.1016/j.ijbiomac.2019.07.070. - DOI - PubMed
    1. Jiang Z., Zhang C., Tang M., Xu B., Wang L., Qian W., He J., Zhao Z., Wu Q., Mu Y., et al. Improving the thermostability of Rhizopus chinensis lipase through site-directed mutagenesis based on B-factor analysis. Front. Microbiol. 2020;11:346. doi: 10.3389/fmicb.2020.00346. - DOI - PMC - PubMed
    1. Pfleger C., Rathi P.C., Klein D.L., Radestock S., Gohlke H. Constraint Network Analysis (CNA): A Python software package for efficiently linking biomacromolecular structure, flexibility, (thermo-) stability, and function. J. Chem. Inf. Model. 2013;53:1007–1015. doi: 10.1021/ci400044m. - DOI - PubMed

LinkOut - more resources