Tài liệu Báo cáo khoa học: Purification and characterization of glutamate N-acetyltransferase

Purification and characterization of glutamate

N-acetyltransferase involved in citrulline accumulation

in wild watermelon

Kentaro Takahara, Kinya Akashi and Akiho Yokota

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Japan

Drought in the presence of strong light is a major

environmental stress that reduces plant productivity

[1]. To adapt to this adverse condition, numerous bio￾chemical and physiological tolerance mechanisms are

expressed in plant cells [2]. One such response involves

accumulation of small organic metabolites, such as

mannitol, proline and glycine betaine, which are collec￾tively referred to as compatible solutes [3]. Compatible

solutes are thought to play important roles in drought

tolerance in plants, acting as mediators of osmotic

adjustment, stabilizers of subcellular structures, and

scavengers of active oxygen radicals [4]. The mecha￾nisms of proline, mannitol, and glycine betaine accu￾mulation are highly regulated through activation of

biosynthesis and ⁄ or suppression of catabolism [3–5].

Wild watermelon plants, which inhabit the Kalahari

Desert, Botswana, exhibit high drought⁄strong-light

stress tolerance [6]. They are able to maintain their

photosynthetic apparatus during prolonged periods of

drought in strong light, suggesting the presence of

Keywords

citrulline; drought/strong-light stress;

glutamate N-acetyltransferase;

thermostability; wild watermelon

Correspondence

A. Yokota, Nara Institute of Science and

Technology, Graduate School of Biological

Sciences, 8916-5 Takayama, Ikoma,

Nara 630-0101, Japan

Fax: +81 743 72 5569

Tel: +81 743 72 5560

E-mail: [email protected]

(Received 12 July 2005, revised 18 August

2005, accepted 23 August 2005)

doi:10.1111/j.1742-4658.2005.04933.x

Citrulline is an efficient hydroxyl radical scavenger that can accumulate at

concentrations of up to 30 mm in the leaves of wild watermelon during

drought in the presence of strong light; however, the mechanism of this

accumulation remains unclear. In this study, we characterized wild water￾melon glutamate N-acetyltransferase (CLGAT) that catalyses the trans￾acetylation reaction between acetylornithine and glutamate to form

acetylglutamate and ornithine, thereby functioning in the first and fifth

steps in citrulline biosynthesis. CLGAT enzyme purified 7000-fold from

leaves was composed of two subunits with different N-terminal amino acid

sequences. Analysis of the corresponding cDNA revealed that these two

subunits have molecular masses of 21.3 and 23.5 kDa and are derived from

a single precursor polypeptide, suggesting that the CLGAT precursor is

cleaved autocatalytically at the conserved ATML motif, as in other glutam￾ate N-acetyltransferases of microorganisms. A green fluorescence protein

assay revealed that the first 26-amino acid sequence at the N-terminus of

the precursor functions as a chloroplast transit peptide. The CLGAT

exhibited thermostability up to 70
C, suggesting an increase in enzyme

activity under high leaf temperature conditions during drought⁄strong-light

stresses. Moreover, CLGAT was not inhibited by citrulline or arginine at

physiologically relevant high concentrations. These findings suggest that

CLGAT can effectively participate in the biosynthesis of citrulline in wild

watermelon leaves during drought⁄strong-light stress.

Abbreviations

AOD, acetylornithine deacetylase; CLGAT, Citrullus lanatus glutamate N-acetyltransferase; DRIP-1, drought-induced polypeptide 1;

DTT, dithiothreitol; GAT, glutamate N-acetyltransfease; GFP, green fluorescence protein.

FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS 5353