Evaluation of the Structure–Function Relationship of SGNH Lipase from Streptomyces rimosus by Site-Directed Mutagenesis and Computational Approach

Filić, Želimira; Bielen, Ana; Šarić, Ela; Ćehić, Mirsada; Crnolatac, Ivo; Tomić, Sanja; Vujaklija, Dušica; Abramić, Marija (2024) Evaluation of the Structure–Function Relationship of SGNH Lipase from Streptomyces rimosus by Site-Directed Mutagenesis and Computational Approach. International Journal of Molecular Sciences, 25 (1). ISSN 1422-0067

PDF - Published Version - article Available under License Creative Commons Attribution. Download (4MB)

Abstract

Streptomyces rimosus extracellular lipase (SrL) is a multifunctional hydrolase belonging to the SGNH family. Here site-directed mutagenesis (SDM) was used for the first time to investigate the functional significance of the conserved amino acid residues Ser10, Gly54, Asn82, Asn213, and His216 in the active site of SrL. The hydrolytic activity of SrL variants was determined using para-nitrophenyl (pNP) esters with C4, C8, and C16 fatty acid chains. Mutation of Ser10, Asn82, or His216, but not Gly54, to Ala abolished lipase activity for all substrates. In contrast, the Asn213Ala variant showed increased enzymatic activity for C8 and C16 pNP esters. Molecular dynamics (MD) simulations showed that the interactions between the long alkyl chain substrate (C16) and Ser10 and Asn82 were strongest in Asn213Ala SrL. In addition to Asn82, Gly54, and Ser10, several new constituents of the substrate binding site were recognized (Lys28, Ser53, Thr89, and Glu212), as well as strong electrostatic interactions between Lys28 and Glu212. In addition to the H bonds Ser10-His216 and His216-Ser214, Tyr11 interacted strongly with Ser10 and His216 in all complexes with an active enzyme form. A previously unknown strong H bond between the catalytically important Asn82 and Gly54 was uncovered, which stabilizes the substrate in an orientation suitable for the enzyme reaction.

Item Type:	Article
Uncontrolled Keywords:	SGNH/GDSL-hydrolase ; streptomyces rimosus ; catalytic efficiency ; catalytic residues ; molecular docking ; molecular dynamics ; protein–substrate interactions ; site-directed mutagenesis ; substrate binding site
Subjects:	NATURAL SCIENCES > Chemistry
Divisions:	Division of Organic Chemistry and Biochemistry Division of Physical Chemistry
Projects:	Project title Project leader Project code Project type Bioprospecting Jadranskog mora- Rozelinda Čož-Rakovac; Verica Dragović-Uzelac; Božidar Šantek; Stela Jokić; Igor Jerković; Sandra Kraljević Pavelić KK.01.1.1.01.0002 EK
Depositing User:	Ana Zečević
Date Deposited:	22 Jul 2024 08:45
URI:	http://fulir.irb.hr/id/eprint/8956
DOI:	10.3390/ijms25010595

Actions (login required)

View Item