Login Register Cart 0
Generic placeholder image

Protein & Peptide Letters

Editor-in-Chief

ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Research Article

Effect of Propeptide Mutations on the Directed Evolution of Rhizomucor miehei Lipase

Author(s): Jue Wang*, Rong Bai, Na Wu, Yumin Zhang and Lili Hu

Volume 29, Issue 4, 2022

Published on: 14 April, 2022

Page: [360 - 369] Pages: 10

DOI: 10.2174/0929866529666220314105130

Price: $65

Abstract

Background: A series of mutants of Rhizomucor miehei lipase (RML) screened through four rounds of directed evolution were studied. Mutants' triglyceride hydrolysis activity was assessed, and their genes were sequenced. Results showed that mutations in the propeptide can improve the activity of RML during evolution. Two parts of propeptide (wild-type and mutant) and mature region were connected by molecular simulation technology.

Methods: The spatial structure of the most positive mutants containing the mutations in the propeptide was mainly characterized by the increase in the opening angle of the lid structure in the mature region of RML, the enhancement of the hydrophobicity of the active center, and the triad of the active center shifted outward.

Results: The three indexes above explain the mechanism of propeptide mutations on the activity change of the target protein. In addition, statistical analysis of all the mutants screened in directed evolution showed that: (1) most of the mutants with increased activity contained mutations of the propeptide, (2) in the later stage of directed evolution, the number of active mutants decreased gradually, and the mutations of inactivated protein mainly occurred in the mature region, and (3) in the last round of directed evolution, the mutations distributed in the propeptide improved the mutant activity further. The results showed that the propeptide reduced RML's evolutionary pressure and delayed the emergence of the evolutionary platform.

Conclusion: These findings reveal the role of propeptide in the evolution of RML and provide strategies for the molecular transformation of other lipases.

Keywords: Lipase, propeptide, activity, directed evolution, mutations, lid structure, active center.

« Previous
Graphical Abstract

[1]
Rodrigues, R.C.; Fernandez-Lafuente, R. Lipase from Rhizomucor miehei as an industrial biocatalyst in chemical process. J. Mol. Catal., B Enzym., 2010, 64, 1-22.
[http://dx.doi.org/10.1016/j.molcatb.2010.02.003]
[2]
Cai, H.Y.; Shen, L.Z.; Zhao, M.J.; Li, Y.; Mao, J.W.; Feng, F.Q. The recombinant expression and Kex2 cleavage site modification of Rhizomucor miehei lipase in Pichia pastoris. J. Chinese Ins. Food Sci. Technol., 2019, 4, 85-91.
[http://dx.doi.org/10.16429/j.1009-7848.2019.04.010]
[3]
Derewenda, Z.S.; Derewenda, U. Relationships among serine hydrolases: Evidence for a common structural motif in triacylglyceride lipases and esterases. Biochem. Cell Biol., 1991, 69(12), 842-851.
[http://dx.doi.org/10.1139/o91-125] [PMID: 1818588]
[4]
Boel, E.; Huge-Jensen, B.; Christensen, M.; Thim, L.; Fiil, N.P. Rhizomucor miehei triglyceride lipase is synthesized as a precursor. Lipids, 1988, 23(7), 701-706.
[http://dx.doi.org/10.1007/BF02535672] [PMID: 3419283]
[5]
Wang, J.; Wang, D.; Wang, B.; Mei, Z.H.; Liu, J.; Yu, H.W. Enhanced activity of Rhizomucor miehei lipase by directed evolution with simultaneous evolution of the propeptide. Appl. Microbiol. Biotechnol., 2012, 96(2), 443-450.
[http://dx.doi.org/10.1007/s00253-012-4049-5] [PMID: 22584429]
[6]
Wang, J. Enhanced the activity and thermostability of Rhizomucor miehei Lipase by directed evolution; Zhe Jiang University: China, 2012.
[7]
Takahashi, S.; Ueda, M.; Tanaka, A. Function of the prosequence for in vivo folding and secretion of active Rhizopus oryzae lipase in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol., 2001, 55(4), 454-462.
[http://dx.doi.org/10.1007/s002530000537] [PMID: 11398926]
[8]
Samuelson, P.; Cano, F.; Robert, A.; Ståhl, S. Engineering of a Staphylococcus carnosus surface display system by substitution or deletion of a Staphylococcus hyicus lipase propeptide. FEMS Microbiol. Lett., 1999, 179(1), 131-139.
[http://dx.doi.org/10.1111/j.1574-6968.1999.tb08718.x] [PMID: 10481097]
[9]
Wang, Z.; Lv, P.; Luo, W.; Yuan, Z.; He, D. Expression in Pichia pastoris and characterization of Rhizomucor miehei lipases containing a new propeptide region. J. Gen. Appl. Microbiol., 2016, 62(1), 25-30.
[http://dx.doi.org/10.2323/jgam.62.25] [PMID: 26923128]
[10]
Luo, W.; He, D.; Fu, J.Y.; Lv, P.M. Effect of propeptide variation on properties of Rhizomucor miehei lipase. J. Biobased Mater. Bioenergy, 2018, 12, 330-338.
[http://dx.doi.org/10.1166/jbmb.2018.1786]
[11]
Chen, Y.J.; Inouye, M. The intramolecular chaperone-mediated protein folding. Curr. Opin. Struct. Biol., 2008, 18(6), 765-770.
[http://dx.doi.org/10.1016/j.sbi.2008.10.005] [PMID: 18973809]
[12]
Nagayama, M.; Maeda, H.; Kuroda, K.; Ueda, M. Mutated intramolecular chaperones generate high-activity isomers of mature enzymes. Biochemistry, 2012, 51(17), 3547-3553.
[http://dx.doi.org/10.1021/bi3001159] [PMID: 22482366]
[13]
Boon, L.; Ugarte-Berzal, E.; Vandooren, J.; Opdenakker, G. Protease propeptide structures, mechanisms of activation, and functions. Crit. Rev. Biochem. Mol. Biol., 2020, 55(2), 111-165.
[http://dx.doi.org/10.1080/10409238.2020.1742090] [PMID: 32290726]
[14]
Demidyuk, I.V.; Shubin, A.V.; Gasanov, E.V.; Kostrov, S.V. Propeptides as modulators of functional activity of proteases. Biomol. Concepts, 2010, 1(3-4), 305-322.
[http://dx.doi.org/10.1515/bmc.2010.025] [PMID: 25962005]
[15]
Saify Nabiabad, H.; Amini, M.; Kianersi, F. Ipomoea batatas: Papain propeptide inhibits cysteine protease in main plant parasites and enhances resistance of transgenic tomato to parasites. Physiol. Mol. Biol. Plants, 2019, 25(4), 933-943.
[http://dx.doi.org/10.1007/s12298-019-00675-3] [PMID: 31402817]
[16]
Moroz, O.V.; Blagova, E.; Reiser, V.; Saikia, R.; Dalal, S.; Jørgensen, C.I.; Bhatia, V.K.; Baunsgaard, L.; Andersen, B.; Svendsen, A.; Wilson, K.S. Novel inhibitory function of the Rhizomucor miehei lipase propeptide and three-dimensional structures of its complexes with the enzyme. ACS Omega, 2019, 4(6), 9964-9975.
[http://dx.doi.org/10.1021/acsomega.9b00612] [PMID: 31460089]
[17]
Miled, N.; De Caro, A.; De Caro, J.; Verger, R. A conformational transition between an open and closed form of human pancreatic lipase revealed by a monoclonal antibody. Biochim. Biophys. Acta, 2000, 1476(2), 165-172.
[http://dx.doi.org/10.1016/S0167-4838(99)00288-5] [PMID: 10669782]
[18]
Derewenda, Z.S.; Derewenda, U.; Dodson, G.G. The crystal and molecular structure of the Rhizomucor miehei triacylglyceride lipase at 1.9 A resolution. J. Mol. Biol., 1992, 227(3), 818-839.
[http://dx.doi.org/10.1016/0022-2836(92)90225-9] [PMID: 1404390]
[19]
Pawar, S.S.; Rohane, S.H. Review on discovery studio: An important tool for molecular docking. Asian J. Res. Chem, 2021, 14, 1-3.
[http://dx.doi.org/10.5958/0974-4150.2021.00014.6]
[20]
Wang, D.; Wang, J.; Wang, B.; Yu, H.W. A new and efficient colorimetric high-throughput screening method for triacylglycerol lipase directed evolution. J. Mol. Catal., B Enzym., 2012, 82, 18-23.
[http://dx.doi.org/10.1016/j.molcatb.2012.05.021]
[21]
Cai, H.; Zhao, M.; Li, Y.; Mao, J.; Cai, C.; Feng, F. Pentapeptide prosequence enhances expression and structure folding of recombinant Thermomyces lanuginosus lipase in Pichia pastoris. Protein Pept. Lett., 2017, 24(7), 676-681.
[http://dx.doi.org/10.2174/0929866524666170621100431] [PMID: 28641563]
[22]
Luo, W. Allosteric regulation and its mechanism of the propeptide on Rhizomucor miehei lipase; South China Agricultural University: China, 2018.
[23]
Zha, D.; Zhang, H.; Zhang, H.; Xu, L.; Yan, Y. N-terminal transmembrane domain of lipase LipA from Pseudomonas protegens Pf-5: A must for its efficient folding into an active conformation. Biochimie, 2014, 105, 165-171.
[http://dx.doi.org/10.1016/j.biochi.2014.07.007] [PMID: 25038570]

Rights & Permissions Print Cite
Article Metrics
24
1
© 2024 Bentham Science Publishers | Privacy Policy