Truncation mutants highlight a critical role for the N- and C-termini of the Spirulina Δ6 desaturase in determining regioselectivity
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Author list: Kurdrid P., Sirijuntarut M., Subudhi S., Cheevadhanarak S., Hongsthong A.
Publisher: Springer
Publication year: 2008
Journal: Molecular Biotechnology (1073-6085)
Volume number: 38
Issue number: 3
Start page: 203
End page: 209
Number of pages: 7
ISSN: 1073-6085
eISSN: 1559-0305
Languages: English-Great Britain (EN-GB)
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Abstract
The results of our previous study on heterologous expression in Escherichia coli of the gene desD, which encodes Spirulina Δ6 desaturase, showed that co-expression with an immediate electron donor-either cytochrome b 5 or ferredoxin-was required for the production of GLA (γ-linolenic acid), the product of the reaction catalyzed by Δ6 desaturase. Since a system for stable transformation of Spirulina is not available, studies concerning Spirulina-enzyme characterization have been carried out in heterologous hosts. In this present study, the focus is on the role of the enzyme's N- and C-termini, which are possibly located in the cytoplasmic phase. Truncated enzymes were expressed in E. coli by employing the pTrcHisA expression system. The truncation of the N- and C-terminus by 10 (N10 and C10) and 30 (N30 and C30) amino acids, respectively, altered the enzyme's regioselective mode from one that measures from a preexisting double bond to that measuring from the methyl end of the substrate. © 2007 Humana Press Inc.
Keywords
C-terminal truncation, Desaturase, fatty acid desaturation, N-terminal truncation, Spirulina platensis
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