Acetohydroxy acid synthase I of Escherichia coli: purification and properties

Abstract

Several properties of the three acetohydroxy acid synthases of Escherichia coli have been compared in crude extracts. The three enzymes can be readily distinguished from each other. Acetohydroxy acid synthase I, the product of the iluB gene, has been purified to near homogeneity. The purification was made possible by the fact that the enzyme was maintained in buffers of a high ionic strength or in buffers containing glycerol. Density gradient centrifugation studies indicated that the enzyme exists as a dimer of subunits of similar (60,000) molecular weight in buffers containing glycerol with or without two of the cofactors, Mg2" and thiamine diphosphate. When flavine adenine dinucleotide was added along with Mg2+ and thiamine diphosphate, an increase in the rate of sedimentation occurred that was thought to be due to a rapid tetramer-dimer interconversion. The addition of pyruvate, the substrate, along with the three cofactors, resulted in a further increase in sedimentation rate, due presumably to an increase in the tetramer-to-dimer ratio. The addition of valine to the complete system resulted in maintenance of the enzyme in the dimeric state concomitant with inhibition of enzyme activity. iWllO (valine resistance) mutations. Finally, acetohydroxy acid synthase III was shown to be the product of a locus near the leu operon which was proposed to contain two genes, ilvH and ilvI. However, owing to the limited evidence for two separate genes, the locus will here be referred to as the iluHI gene. The third isozyme was recently shown by DeFelice and Levinthal (9) to be strongly repressed by leucine alone.A clear understanding of this gene-enzyme relationship will be dependent not only upon a more careful genetic analysis than has been performed until now but also upon a distinction 846 on July 16, 2020 by guest

Acetohydroxyacid synthase from Claviceps purpurea: Partial purification and characterization

“…This value is comparable to those found in other acetolactate forming systems (GRIMMINGER andUMBARGER 1979, SCHLOSS el ul. 1985).…”

Physical and kinetic properties of acetohydroxyacid synthase from wheat leaves

“…2). The 57and 58-kDa polypeptides of AHAS from wheat were similar in size to the single subunit of AHAS isolated from barley (Dumer and Boger 1988) and to the large subunit of AHAS from bacteria (Grimminger and Umbarger 1979, Eoyang and Silverman 1984, Schloss et al 1985. In contrast, a single polypeptide of molecular mass of approximately 65 kDa has been identified in immunoblots of crude extracts from a wide range of plant tissues using monoclonal antibodies raised against synthetic peptides as immunogen, or polyclonal antibodies raised agairtst an Arabidopsis AHAS fusion protein expressed in E. coli (Singh et al 1991a,b).…”

“…1988a). Most reports of the native molecular mass of AHAS from bacteria fall in the range between 130 and 160 kDa (Grimminger and Umbarger 1979, Eoyang and Silverman 1984, Schloss et al 1985, Alexander-Caudle et al 1990), although 68-, 140-and 500-kDa forms of the enzyme were reported in Neurospora crassa (Tanaka and Kuwana 1984).…”

Bacterial acetohydroxyacid synthase and its inhibitors – a summary of their structure, biological activity and current status

“…AHAS expression and purification from native sources has been described in detail previously by Duggleby et al [1]. Enterobacterial isozymes are more easily purified from native sources than eukaryotic AHASs [34][35][36]. The respective expression of the three isozymes is regulated in different ways in enterobacteria.…”