Inhibition of Glutamine Synthetase from Pea by Tabtoxinine-β-lactam

Abstract

Tabtoxinine-f-lactam, a hydrolytic product of tabtoxin produced by Pseudoonas syringae pv. tabaci, apparently inactvates pea seed glutamine synthetase. Inhibition of the enzyme's initial velocity is linear over a range of 0.5 to 5 minhmmolar tabtoxinne-/-lactam in the presence of 10 m_mlar glutamate. A method for the purification of glutamine synthetase from dried peas is presented which gives a 30% yield with a 2,000-fold Increase in specific activity. A method for obtaining highly puied tabtoxinine-f8-lactam and tabtoxin in good yields Is also presented. The authenticity and purity of tabtoxinine*lactam and tabtoxin were verified by chromatography, biological activity, and 1H and 13C nuclear magnetic resonance spectroscopy.The phytopathogenic bacterium Pseudomonas syringae pv. tabaci and certain other closely related pathovars produce a toxin which induces chlorosis in plants (5,11,12,16). This toxin, called tabtoxin, is a dipeptide composed of either threonine or serine and the novelf,-lactam-containing amino acid, tabtoxinine-,8-lactam (2-amino-4-[3-hydroxy-2-oxo-azacyclobutan-3-yl]-butanoic acid).Originally, it was hypothesized that chlorosis was caused by a rapid buildup of ammonium, resulting from the inhibition of GS' (EC 6.3.1.2) by tabtoxin. Semipurified tabtoxin did, in fact, inhibit the activity of crude GS from pea (14). This hypothesis was also supported by the following observations: (a) MSO, which was already known to inhibit GS, also caused chlorosis and ammonium accumulation (7, 10); (b) tabtoxin-and MSO-induced chlorosis and ammonium accumulation are light-dependent reactions (3) which can be inhibited by dichlorodimethyl phenylurea (Durbin, unpublished); and (c) glutamine negated the inhibitory effects of both MSO and tabtoxin in several biological systems (2). Preliminary studies using purified materials failed to show inhibition of GS by tabtoxin (19). This has been clarified by the present study and the recent observation that unidentified peptidases in tobacco will hydrolyze tabtoxin to yield tabtoxinine-,flactam (19). Inasmuch as this compound can also cause chlorosis and, more important, appeared to inhibit GS, we postulated that chlorosis development via an inhibition of this enzyme depended upon the generation of tabtoxinine-,B-lactam.This report establishes that purified tabtoxinine-fl-lactam, but not tabtoxin, inhibits purified GS from pea by apparently irreversibly inactivating the enzyme. A simple, rapid procedure for the purification of tabtoxin and tabtoxinine-f,-lactam is also reported.'Abbreviations: GS, glutamine synthetase; MSO, L-methionine-S-sulfoximine; NMR, nuclear magnetic resonance. MATERIALS AND METHODSEnzyme Purification. Commercially obtained, dried split peas (1 kg) were milled to a fine powder and soaked 24 h at 4°C in 2 L 20 mm imidazole (pH 7.5), 10 mM MgC12, and 1 mM ,B-mercaptoethanol. The crude enzyme preparation was clarified by centrifugation (14,000g for 30 min), and a fraction containing the enzyme activity was precipitated by adjusting the pH to ...

Novel type of adenylyl cyclase participates in tabtoxinine-β-lactam-induced cell death and occurrence of wildfire disease inNicotiana benthamiana

“…26 P. syringae pv tabaci 6605 was cultured in PY medium containing 20 µg/ mL rifampicin. 27 Purification of TβL was performed according to the method described by Tomas et al 28 Bacterial suspension, l-methionine sulfoximine (MSX; Funakoshi) and TβL were infiltrated into N. benthamiana leaves as described previously. 9 Isolation of RNA and cDNA synthesis Total RNA was isolated from N. benthamiana leaves with RNAiso (Takara), and RNA samples were treated with DNase I (RNase-free; Takara) to degrade contaminating genomic DNA as described previously.…”

Role of glutamine synthetase adenylylation in the self-protection of Pseudomonas syringae subsp. "tabaci" from its toxin, tabtoxinine-beta-lactam

“…T-,-L was obtained by the method of Thomas et al (17). Attempts to recover GS activity by dialysis after inactivation were done by the methods of Maurizi and Ginsburg (7).…”

“…The physiological target in plants, glutamine synthetase (GS) (EC 6.3.2.1.) (5,6,17) is irreversibly inhibited by T-P-L (6,17). A preliminary study by Thomas and Durbin (16) showed that GS from subsp.…”

Virulence strategies of phytopathogenic bacteria and their role in plant disease pathogenesis

“…Some are polypeptids, glycoproteins others are secondary metabolites that are required as virulence factors in plant disease (Alfano and Collmer, 2004). These toxins acts by using diverse mechanisms from modulating and suppressing plant defense response to alteration and inhibition of normal host cellular metabolic process (Thomas et al, 1983). These toxins act also directly on the expression and development of disease symptoms.…”

“…These toxins act also directly on the expression and development of disease symptoms. Among the most well studied pathotoxins known also as phytotoxins include syringomycins, syringopeptins, tabtoxins, phaseolotoxins and coranotine described particularly in P. syringae pathovars (Thomas et al, 1983).…”