Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 326091
Title Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specifity.
Author(s) Tamer, M.K. el; Lucker, J.; Bosch, D.; Verhoeven, H.A.; Verstappen, F.W.A.; Schwab, W.; Tunen, A.J. van; Voragen, A.G.J.; Maagd, R.A. de; Bouwmeester, H.J.
Source Archives of Biochemistry and Biophysics 411 (2003). - ISSN 0003-9861 - p. 196 - 203.
DOI https://doi.org/10.1016/S0003-9861(02)00711-7
Department(s) Food Chemistry
BIOS Applied Metabolic Systems
PRI Bioscience
VLAG
Publication type Refereed Article in a scientific journal
Publication year 2003
Keyword(s) site-directed mutagenesis - 5-epi-aristolochene synthase - trichodiene synthase - linalool synthase - germacrene-a - biosynthesis - expression - biology
Abstract Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a P-pinene synthase named Cl(-)betaPINS, a gamma-terpinene synthase named ClgammaTS, and two limonene synthases named Cl(+)LIMS1 and Cl(+)LIMS2 were recently isolated [J. Lucker et al., Eur. J. Biochem. 269 (2002) 3160]. The aim of our work in this study was to identify domains within these monoterpene synthase enzymes determining the product specificity. Domain swapping experiments between Cl(-)betaPINS and ClgammaTS and between Cl(+)LIMS2 and ClyTS were conducted. We found that within the C-terminal domain of these monoterpene synthases, a region comprising 200 amino acids, of which 41 are different between Cl(-)betaPINS and ClgammaTS, determines the specificity for the formation of P-pinene or gamma-terpinene, respectively, while another region localized further downstream is required for a chimeric enzyme to yield products in the same ratio as in the wild-type ClgammaTS. For Cl(+)LIMS2, the two domains together appear to be sufficient for its enzyme specificity, but many chimeras were inactive probably due to the low homology with ClyTS. Molecular modeling was used to further pinpoint the amino acids responsible for the differences in product specificity of ClyTS and Cl(-)betaPINS. (C) 2003 Elsevier Science (USA). All rights reserved.
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