title: Truncation mutants highlight a critical role for the N- and C-termini of the Spirulina Delta(6) desaturase in determining regioselectivity.
abstract: 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.
authors.affiliation: 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.
authors.forename: Pavinee
authors.initials: P
authors.lastname: Kurdrid
authors.forename: Matura
authors.initials: M
authors.lastname: Sirijuntarut
authors.forename: Sanjukta
authors.initials: S
authors.lastname: Subudhi
authors.forename: Supapon
authors.initials: S
authors.lastname: Cheevadhanarak
authors.forename: Apiradee
authors.initials: A
authors.lastname: Hongsthong
mesh_terms: 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
chemical_list: D001426:Bacterial Proteins; D005227:Fatty Acids; D011994:Recombinant Proteins; D050602:Linoleoyl-CoA Desaturase