TY - JOUR
T1 - Characterization and cloning of a stearoyl/oleoyl specific fatty acyl-acyl carrier protein thioesterase from the seeds of Madhuca longifolia (latifolia)
AU - Ghosh, Santosh K.
AU - Bhattacharjee, Ashish
AU - Jha, Jyoti K.
AU - Mondal, Ashis K.
AU - Maiti, Mrinal K.
AU - Basu, Asitava
AU - Ghosh, Dolly
AU - Ghosh, Sudhamoy
AU - Sen, Soumitra K.
N1 - Funding Information:
Partial grant support to SKS from the Department of Biotechnology, Govt. of India, for this study is gratefully acknowledged. A research fellowship to JKJ from the University Grants Commission, New Delhi, is also acknowledged herewith. Thanks are also due to Dr K.D. Chapman for providing the E. coli strain K27fadD88 and to Dr D.R. Mal of Department of Chemistry, IIT, Kharagpur, for the intellectual support rendered during the course of the study. Technical help received from Mr Anirudhya Aich of Bose Institute, Calcutta, Mr Ashok Jana and Ms Dolly Jana of the laboratory are gratefully acknowledged.
PY - 2007/12
Y1 - 2007/12
N2 - Deposition of oleate, stearate and palmitate at the later stages of seed development in Mahua (Madhuca longifolia (latifolia)), a tropical non-conventional oil seed plant, has been found to be the characteristic feature of the regulatory mechanism that produces the saturated fatty acid rich Mahua seed fat (commonly known as Mowrah fat). Although, the content of palmitate has been observed to be higher than that of stearate at the initial stages of seed development, it goes down when the stearate and oleate contents consistently rise till maturity. The present study was undertaken in order to identify the kind of acyl-ACP thioesterase(s) that drives the characteristic composition of signature fatty acids (oleate 37%, palmitate 25%, stearate 23%, linoleate 12.5%) in its seed oil at maturity. The relative Fat activities in the crude protein extracts of the matured seeds towards three thioester substrates (oleoyl-, stearoyl- and palmitoyl-ACP) have been found to be present in the following respective ratio 100:31:8. Upon further purification of the crude extract, the search revealed the presence of two partially purified thioesterases: a long-chain oleoyl preferring house-keeping LC-Fat and a novel stearoyl-oleoyl preferring SO-Fat. The characteristic accumulation of oleate and linoleate in the M. latifolia seed fat is believed to be primarily due to the thioesterase activity of the LC-Fat or MlFatA. On the other hand, the SO-Fat showed almost equal substrate specificity towards stearoyl- and oleoyl-ACP, when its activity towards palmitoyl-ACP compared to stearoyl-ACP was only about 12%. An RT-PCR based technique for cloning of a DNA fragment from the mRNA pool of the developing seed followed by nucleotide sequencing resulted in the identification of a FatB type of thioesterase gene (MlFatB). This gene was found to exist as a single copy in the mother plant genome. Ectopic expression of this MlFatB gene product in E. coli strain fadD88 further proved that it induced a higher level of accumulation of both stearic and oleic acids when compared to the negative control line that did not contain this MlFatB gene. It also indicated that SO-Fat indeed is the product of the MlFatB gene present in the maturing seeds of M. latifolia in nature. Additionally, a predicted 3D-structure for MlFatB protein has been developed through use of bioinformatics tools.
AB - Deposition of oleate, stearate and palmitate at the later stages of seed development in Mahua (Madhuca longifolia (latifolia)), a tropical non-conventional oil seed plant, has been found to be the characteristic feature of the regulatory mechanism that produces the saturated fatty acid rich Mahua seed fat (commonly known as Mowrah fat). Although, the content of palmitate has been observed to be higher than that of stearate at the initial stages of seed development, it goes down when the stearate and oleate contents consistently rise till maturity. The present study was undertaken in order to identify the kind of acyl-ACP thioesterase(s) that drives the characteristic composition of signature fatty acids (oleate 37%, palmitate 25%, stearate 23%, linoleate 12.5%) in its seed oil at maturity. The relative Fat activities in the crude protein extracts of the matured seeds towards three thioester substrates (oleoyl-, stearoyl- and palmitoyl-ACP) have been found to be present in the following respective ratio 100:31:8. Upon further purification of the crude extract, the search revealed the presence of two partially purified thioesterases: a long-chain oleoyl preferring house-keeping LC-Fat and a novel stearoyl-oleoyl preferring SO-Fat. The characteristic accumulation of oleate and linoleate in the M. latifolia seed fat is believed to be primarily due to the thioesterase activity of the LC-Fat or MlFatA. On the other hand, the SO-Fat showed almost equal substrate specificity towards stearoyl- and oleoyl-ACP, when its activity towards palmitoyl-ACP compared to stearoyl-ACP was only about 12%. An RT-PCR based technique for cloning of a DNA fragment from the mRNA pool of the developing seed followed by nucleotide sequencing resulted in the identification of a FatB type of thioesterase gene (MlFatB). This gene was found to exist as a single copy in the mother plant genome. Ectopic expression of this MlFatB gene product in E. coli strain fadD88 further proved that it induced a higher level of accumulation of both stearic and oleic acids when compared to the negative control line that did not contain this MlFatB gene. It also indicated that SO-Fat indeed is the product of the MlFatB gene present in the maturing seeds of M. latifolia in nature. Additionally, a predicted 3D-structure for MlFatB protein has been developed through use of bioinformatics tools.
KW - FatB cDNA
KW - Fatty acyl-acyl carrier protein (ACP) thioesterase
KW - Madhuca longifolia (latifolia)
KW - Mowrah fat
KW - Oleoyl-ACP thioesterase
KW - Stearoyl-ACP thioesterase
UR - http://www.scopus.com/inward/record.url?scp=36248961691&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=36248961691&partnerID=8YFLogxK
U2 - 10.1016/j.plaphy.2007.09.003
DO - 10.1016/j.plaphy.2007.09.003
M3 - Article
C2 - 17977002
AN - SCOPUS:36248961691
SN - 0981-9428
VL - 45
SP - 887
EP - 897
JO - Plant Physiology and Biochemistry
JF - Plant Physiology and Biochemistry
IS - 12
ER -