Molecular mechanisms underlying catalytic activity of delta 6 desaturase from Glossomastix chrysoplasta and Thalassiosira pseudonana

Haisu Shi,史海 粟,Rina Wu,乌日 娜,Yan Zheng,郑 艳,Xiqing Yue,岳喜 庆

doi:10.1194/jlr.m079806

Haisu Shi, 史海粟 + Show 6 more

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https://doi.org/10.1194/jlr.m079806

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Journal: Journal of Lipid Research	Publication Date: Jan 1, 2018
Citations: 12	License type: cc-by

Affiliation: Shenyang Agricultural University

Abstract

Delta 6 desaturase (FADS2) is a critical bifunctional enzyme required for PUFA biosynthesis. In some organisms, FADS2s have high substrate specificity, whereas in others, they have high catalytic activity. Previously, we analyzed the molecular mechanisms underlying high FADS2 substrate specificity; in this study, we assessed those underlying the high catalytic activity of FADS2s from Glossomastix chrysoplasta and Thalassiosira pseudonana. To understand the structural basis of this catalytic activity, GcFADS2 and TpFADS2 sequences were divided into nine sections, and a domain-swapping approach was applied to examine the role of each section in facilitating the catalytic activity of the overall protein. The results revealed two regions essential to this process: one that extends from the end of the fourth to the beginning of the fifth cytoplasmic transmembrane domain, and another that includes the C-terminal region that occurs after the sixth cytoplasmic transmembrane domain. Based on the domain-swapping analyses, the amino acid residues at ten sites were identified to differ between the GcFADS2 and TpFADS2 sequences, and therefore further analyzed by site-directed mutagenesis. T302V, S322A, Y375F, and M384S/M385 substitutions in TpFADS2 significantly affected FADS2 catalytic efficiency. This study offers a solid basis for in-depth understanding of catalytic efficiency of FADS2.

Highlights

Delta 6 desaturase (FADS2) is a critical bifunctional enzyme required for PUFA biosynthesis
After initial denaturation at 95°C for 5 min, PCR amplification was performed in 30 cycles of 50 s at 95°C, 50 s at 58°C, and Cloning and characterization of TpFADS2 and GcFADS2 To characterize the catalytic activities of TpFADS2 and
TpFADS2 was shown to convert LA/ALA: 18:2 (LA) and -linolenic acid (ALA) at a rate of 65.6 ± 1.8% and 77.6 ± 1.9%, respectively, while GcFADS2 only catalyzed the conversion of LA and ALA at a rate of 5.6 ± 0.8% and 7.6 ± 0.6%, respectively (Table 1)

Summary

Introduction

Delta 6 desaturase (FADS2) is a critical bifunctional enzyme required for PUFA biosynthesis. FADS2s have high substrate specificity, whereas in others, they have high catalytic activity. We analyzed the molecular mechanisms underlying high FADS2 substrate specificity; in this study, we assessed those underlying the high catalytic activity of FADS2s from Glossomastix chrysoplasta and Thalassiosira pseudonana. To understand the structural basis of this catalytic activity, GcFADS2 and TpFADS2 sequences were divided into nine sections, and a domain-swapping approach was applied to examine the role of each section in facilitating the catalytic activity of the overall protein. Based on the domain-swapping analyses, the amino acid residues at ten sites were identified to differ between the GcFADS2 and TpFADS2 sequences, and further analyzed by sitedirected mutagenesis. This study offers a solid basis for indepth understanding of catalytic efficiency of FADS2.—Shi, H., R. Yue. Molecular mechanisms underlying catalytic activity of delta 6 desaturase from Glossomastix chrysoplasta and Thalassiosira pseudonana.

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