In an attempt to rationalize the relationship between structure and substrate selectivity of glycerol-3-phosphate acyltransferase (GPAT, 1AT, EC 2.3.1.15) we have cloned a number of cDNAs into the PET overexpression system using a PCR-based approach. Following assay of the recombinant enzyme we noted that the substrate selectivity of the squash (Cucurbita moschata) enzyme had altered dramatically. This form of GPAT has now been crystallized and its full three-dimensional structure elucidated. Since we now have two forms of the enzyme that display different substrate selectivities this should provide a powerful tool to determine the basis of the selectivity changes. Kinetic and structural analyses are currently being performed to rationalize the changes which have taken place.
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Conference Article|
December 01 2000
Mutagenesis of squash (Cucurbita moschata) glycerol-3-phosphate acyltransferase (GPAT) to produce an enzyme with altered substrate selectivity
M. W. Hayman;
M. W. Hayman
*Department of Biological Sciences, University of Durham, South Road, Durham DH1 3LE, U.K.
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T. Fawcett;
T. Fawcett
*Department of Biological Sciences, University of Durham, South Road, Durham DH1 3LE, U.K.
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T. F. Schierer;
T. F. Schierer
*Department of Biological Sciences, University of Durham, South Road, Durham DH1 3LE, U.K.
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J. W. Simon;
J. W. Simon
*Department of Biological Sciences, University of Durham, South Road, Durham DH1 3LE, U.K.
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J. T. M. Kroon;
J. T. M. Kroon
*Department of Biological Sciences, University of Durham, South Road, Durham DH1 3LE, U.K.
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J. S. Gilroy;
J. S. Gilroy
*Department of Biological Sciences, University of Durham, South Road, Durham DH1 3LE, U.K.
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D. W. Rice;
D. W. Rice
†Department of Molecular Biology, University of Sheffield, Sheffield S10 2TN, U.K.
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J. Rafferty;
J. Rafferty
†Department of Molecular Biology, University of Sheffield, Sheffield S10 2TN, U.K.
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A. P. Turnbull;
A. P. Turnbull
†Department of Molecular Biology, University of Sheffield, Sheffield S10 2TN, U.K.
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S. E. Sedelnikova;
S. E. Sedelnikova
†Department of Molecular Biology, University of Sheffield, Sheffield S10 2TN, U.K.
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A. R. Slabas
A. R. Slabas
1
*Department of Biological Sciences, University of Durham, South Road, Durham DH1 3LE, U.K.
1To whom correspondence should be addressed (e-mail a.r.slabas@durham.ac.uk)
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Publisher: Portland Press Ltd
Received:
June 28 2000
Online ISSN: 1470-8752
Print ISSN: 0300-5127
© 2000 Biochemical Society
2000
Biochem Soc Trans (2000) 28 (6): 680–681.
Article history
Received:
June 28 2000
Citation
M. W. Hayman, T. Fawcett, T. F. Schierer, J. W. Simon, J. T. M. Kroon, J. S. Gilroy, D. W. Rice, J. Rafferty, A. P. Turnbull, S. E. Sedelnikova, A. R. Slabas; Mutagenesis of squash (Cucurbita moschata) glycerol-3-phosphate acyltransferase (GPAT) to produce an enzyme with altered substrate selectivity. Biochem Soc Trans 1 December 2000; 28 (6): 680–681. doi: https://doi.org/10.1042/bst0280680
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