Applied Microbiology and Biotechnology

, Volume 66, Issue 1, pp 74–84

Mutation study of conserved amino acid residues of Spirulina Δ6-acyl-lipid desaturase showing involvement of histidine 313 in the regioselectivity of the enzyme

  • Apiradee Hongsthong
  • Sanjukta Subudhi
  • Matura Sirijuntarat
  • Supapon Cheevadhanarak
Applied Genetics and Molecular Biotechnology

DOI: 10.1007/s00253-004-1655-x

Cite this article as:
Hongsthong, A., Subudhi, S., Sirijuntarat, M. et al. Appl Microbiol Biotechnol (2004) 66: 74. doi:10.1007/s00253-004-1655-x

Abstract

In the cyanobacterium Spirulina platensis, the desaturation process is carried out by three desaturases: the Δ9, Δ12 and Δ6 desaturases, encoded by desC, desA and desD, respectively. The Δ6 desaturase is responsible for the catalysis of linoleic acid, yielding γ-linolenic acid (18:3Δ9,12,6), 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-à-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 the activity of Δ6 desaturase, possibly by forming part of the substrate-binding pocket.

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Apiradee Hongsthong
    • 1
  • Sanjukta Subudhi
    • 1
  • Matura Sirijuntarat
    • 2
  • Supapon Cheevadhanarak
    • 2
  1. 1.Biochemical Engineering and Pilot Plant Research and Development Unit, National Center for Genetic Engineering and BiotechnologyKing Mongkut’s University of Technology–Thonburi (Bangkhuntien)BangkokThailand
  2. 2.School of Bioresources and TechnologyKing Mongkut’s University of Technology–Thonburi (Bangkhuntien)BangkokThailand