Truncation mutants highlight a critical role for the N- and C-termini of the Spirulina Delta(6) desaturase in determining regioselectivity.
The results of our previous study on heterologous expression in Escherichia coli of the gene desD, which encodes Spirulina Delta(6) desaturase, showed that co-expression with an immediate electron donor-either cytochrome b ( 5 ) or ferredoxin-was required for the production of GLA (gamma-linolenic acid), the product of the reaction catalyzed by Delta(6) 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.
Kurdrid
Pavinee
P
Biochemical Engineering and Pilot Plant Research and Development Unit, National Center for Genetic Engineering and Biotechnology, King Mongkut's University of Technology-Thonburi (Bangkhuntien), 83 Moo 8, Thakham, Bangkhuntien, Bangkok, 10150, Thailand.
Sirijuntarut
Matura
M
Subudhi
Sanjukta
S
Cheevadhanarak
Supapon
S
Hongsthong
Apiradee
A
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002849:Chromatography, Gas; D004926:Escherichia coli / Q000235:genetics; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D050602:Linoleoyl-CoA Desaturase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D053545:Spirulina / Q000201:enzymology* / Q000235:genetics; D013237:Stereoisomerism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005227:Fatty Acids; D011994:Recombinant Proteins; D050602:Linoleoyl-CoA Desaturase