Mutation study of conserved amino acid residues of Spirulina delta 6-acyl-lipid desaturase showing involvement of histidine 313 in the regioselectivity of the enzyme

Applied Microbiology and Biotechnology
Apiradee HongsthongSupapon Cheevadhanarak

Abstract

In the cyanobacterium Spirulina platensis, the desaturation process is carried out by three desaturases: the Delta(9), Delta(12) and Delta(6) desaturases, encoded by desC, desA and desD, respectively. The Delta(6) desaturase is responsible for the catalysis of linoleic acid, yielding gamma-linolenic acid (18:3(Delta 9,12,6)), the end-product of the process. In this study, the desD gene was expressed in Escherichia coli using a pTrcHisA expression system. In order to identify the amino acid residues involved in the enzymatic activity, a sequence comparison was performed using various organisms. The alignment revealed three conserved histidine clusters, a number of conserved residues among all listed organisms and a few conserved residues among cyanobacterial species possibly involved in the desaturation activity. A series of site-directed mutations were generated in the desD gene to evaluate the role of these residues vis-a-vis the enzyme function. This approach revealed that: (1) H313 is involved in the regioselectivity of the enzyme, (2) the three histidine clusters together with H313, H315, D138 and E140 are required for enzymatic activity, most likely as providers of the catalytic Fe center and (3) W294 is also essential for...Continue Reading

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Citations

Nov 2, 2013·Applied Microbiology and Biotechnology·Mingxuan WangYong Q Chen
Dec 6, 2005·Molecular Biology Reports·Pavinee KurdridMorakot Tanticharoen
Sep 30, 2008·DNA Research : an International Journal for Rapid Publication of Reports on Genes and Genomes·Masayuki MachidaKatsuya Gomi
Oct 4, 2006·Environmental Microbiology·Patrick D CurtisLawrence J Shimkets
May 23, 2009·FEMS Microbiology Letters·Michael W RingHelge B Bode
Dec 13, 2005·Biochemical and Biophysical Research Communications·Sutthicha Na-RanongSupapon Cheevadhanarak
Aug 9, 2018·Journal of Lipid Research·Diego E SastreSilvia G Altabe
Nov 30, 2012·Plant Biotechnology Journal·Thomas VanherckeAllan G Green
Jul 26, 2017·Zeitschrift Für Naturforschung. C, a Journal of Biosciences·Michal TupecIva Pichová
Jun 17, 2020·Computational and Structural Biotechnology Journal·Aleš BučekIva Pichová
Jan 30, 2013·Biochemical and Biophysical Research Communications·Li-Ying SongZan-Min Hu
Oct 20, 2005·Journal of Bioscience and Bioengineering·Takahiro AbeSakayu Shimizu
Nov 7, 2021·World Journal of Microbiology & Biotechnology·Hang-Qin ZhuYu-Guo Zheng

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