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JXB Advance Access originally published online on March 5, 2009
Journal of Experimental Botany 2009 60(6):1743-1757; doi:10.1093/jxb/erp044
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© 2009 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Biochemical characterization, mitochondrial localization, expression, and potential functions for an Arabidopsis {gamma}-aminobutyrate transaminase that utilizes both pyruvate and glyoxylate

Shawn M. Clark1, Rosa Di Leo1, Preetinder K. Dhanoa2, Owen R. Van Cauwenberghe1 *, Robert T. Mullen2 and Barry J. Shelp1,§

1Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada N1G 2W1
2Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1

§ To whom correspondence should be addressed. E-mail: bshelp{at}uoguelph.ca

{gamma}-Aminobutyrate transaminase (GABA-T) catalyses the breakdown of GABA to succinic semialdehyde. In this report, the previously identified Arabidopsis thaliana (L.) Heyhn GABA-T (AtGABA-T) was characterized in more detail. Full-length AtGABA-T contains an N-terminal 36 amino acid long targeting pre-sequence (36 amino acids) that is both sufficient and necessary for targeting the enzyme to mitochondria. Removal of the pre-sequence encoding this N-terminal targeting domain and co-expression of the resulting truncated AtGABA-T cDNA with the GroES/EL molecular chaperone complex in Escherichia coli yielded good recovery of the soluble recombinant proteins. Activity assays indicated that purified recombinant GABA-T has both pyruvate- and glyoxylate-dependent activities, but cannot utilize 2-oxoglutarate as amino acceptor. Kinetic parameters for glyoxylate- and pyruvate-dependent GABA-T activities were similar, with physiologically relevant affinities. Assays of GABA-T activity in cell-free leaf extracts from wild-type Arabidopsis and two knockout mutants in different genetic backgrounds confirmed that the native enzyme possesses both pyruvate- and glyoxylate-dependent activities. The GABA-T transcript was present throughout the plant, but its expression was highest in roots and increased as a function of leaf development. A GABA-T with dual functions suggests the potential for interaction between GABA metabolism and photorespiratory glyoxylate production.

Key words: Enzyme kinetics, GABA transaminase, gene expression, Glyoxylate, Photorespiration, Recombinant protein, Subcellular localization

* Present address: Lilly Analytical Research Laboratory, Eli Lilly Canada Inc., 3650 Danforth Avenue, Toronto, ON M1N 2E8, Canada

Received 25 November 2008; Revised 31 January 2009 Accepted 4 February 2009


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S. M. Clark, R. Di Leo, O. R. Van Cauwenberghe, R. T. Mullen, and B. J. Shelp
Subcellular localization and expression of multiple tomato {gamma}-aminobutyrate transaminases that utilize both pyruvate and glyoxylate
J. Exp. Bot., July 1, 2009; 60(11): 3255 - 3267.
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