Sulfometuron methyl-sensitive and -resistant acetolactate synthases of the archaebacteria Methanococcus spp

Abstract

The herbicide sulfometuron methyl (SM) inhibited growth of some methanococci. Of 28 strains tested, the growth of 7 was completely inhibited by 0.55 mM SM. Growth of an additional 14 strains was partially inhibited, and the growth of 7 strains was unaffected by this concentration of SM. In some cases, the branched-chain amino acids protected growth. Growth inhibition was correlated with the K, for SM of acetolactate synthase (ALS). For the enzymes from bacteria representative of the sensitive, partially resistant, and resistant methanococci (Methanococcus aeolicus, Methanococcus maripaludis, and Methanococcus voltae, respectively), the Ki for SM was 0.0012, 0.34, and >1.0 mM, respectively. Inhibition was uncompetitive with respect to pyruvate. Based on these observations, ALS appeared to be the major if not the sole site of action of SM in the methanococci. The sensitivity of the ALS from these three methanococci to feedback inhibition by branched-chain amino acids was also quite different. Although all three were sensitive to feedback inhibition by valine, the Ki varied 20-fold, from 0.01 to 0.22 mM. Moreover, only the ALS from M. maripaludis was sensitive to inhibition by leucine, and the Ki was 1.8 mM. The Ki for isoleucine for the ALS from both M. maripaludis and M. voltae was about 0.1 mM. The ALS from M. aeolicus was not inhibited by isoleucine. In other respects, the ALSs from the methanococci were very similar. After dialysis, thiamine pyrophosphate but not FAD and Mg2+ was required for maximal activity, and they were all rapidly inactivated by oxygen. Although the methanococcal ALSs exhibited diverse properties, the range of catalytic and regulatory properties closely resembled those of the eubacterial enzymes.

Purification and characterization of the oxygen-sensitive acetohydroxy acid synthase from the archaebacterium Methanococcus aeolicus

“…While the methanococcal enzymes in the branched-chain amino acid biosynthetic pathway have not been studied in detail, preliminary investigations with partially purified enzymes have demonstrated that the methanococcal AHAS and other enzymes of the pathway resemble the eubacterial and eucaryotic enzymes with regard to their catalytic properties and native molecular weights (35)(36)(37). This result suggests that this pathway is very ancient and that the AHASs from organisms of the three primary domains are homologous.…”

“…AHAS assay. AHAS was measured under anaerobic conditions as described previously (35). The standard assay mixture contained 0.1 M Tricine {N-[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]glycine} (pH 7.5), 50 mM pyruvate, 10.0 mM MgCl2, 0.1 mM TPP, and 0.1 mM FAD.…”

“…Cell extract was prepared as described previously except that 20% (vol/vol) glycerol was added to the PIPES [piperazine-N,N'-bis(2-ethanesulfonate)] buffer, pH 7.5 (35).…”

Cold Shock of a Hyperthermophilic Archaeon: Pyrococcus furiosus Exhibits Multiple Responses to a Suboptimal Growth Temperature with a Key Role for Membrane-Bound Glycoproteins

“…All assays were carried out at 85°C using cell extracts that were prepared as described previously (1). Acetolactate synthase activity was measured by the formation of acetoin (55). Aminoacyl-releasing enzyme (AARE) activity was measured by the release of p-nitroaniline from a tripeptide substrate (19).…”

Inhibitors of Bacillus anthracis acetohydroxyacid synthase

“…The sulfonylureas have been reported to be mixed [27], uncompetitive [28], and nearly competitive inhibitors with respect to pyruvate [29]. IQ (K ii 2.02 M) was an uncompetitive inhibitor for the catalytic subunit of BAntx AHAS.…”