Subunit Structure of Aspartokinase III of Escherichia coli K12

Abstract

The lysine-sensitive aspartokinase I11 of Escherichia coli K12 is composed of subunits of 50000 molecular weight. By determination of terminal residues and inspection of tryptic hydrolysis products, the subunits appear identical. The native enzyme is a dimer of 105000 molecular weight in the presence of 0.15 M KC1 in the absence of lysine. Two types of equilibrium can be evidenced : an association at high ionic strength in the presence of lysine leading to a tetrameric form and a dissociation towards monomer at low ionic strength in the absence of lysine. The two equilibria are dependent on protein concentration.The lysine-sensitive aspartokinase of Escherichia coli K12 is one of the three enzymes catalyzing the phosphorylation of the /?-carboxyl of aspartate in these bacteria [1,2]. Lysine is the feed-back inhibitor of this activity [l] The protein from E. coli K12 has been purified to homogeneity [6 -81 ; molecular weights of 100 000 [6], 177000 [7] and 130000 [S] have been proposed for the native enzyme. von Dippe et al.[9] reported a subunit molecular weight of 39000 for the E . coli B enzyme, the native enzyme undergoing a dimertetramer transition in the presence of lysine and Mg2+.As this paper was to be submitted for publication a paper by Niles and Westhead appeared [lo] where they proposed for aspartokinase I11 from E. coli K12 a tetrameric structure with 48000-molecular-weight subunits ; the tetramer was obtained in 0.1 M KC1 and 1 mM lysine at high protein concentration. A variable quaternary structure of the enzyme is described and proposed to be related to the growth phase and to an adenylation of the enzyme [lo, 111 : one form is isolated in the tetrameric state, while the second is a stable dimer showing no aggregation process. We present results for the subunit structure of E . coli K12 aspartokinase I11 in agreement with the one proposed by Niles and Westhead [lo]. I n addition, we show that the enzyme subunits are identical, that a dimeric form is observed in the presence of KC1 without lysine and that this dimer either aggregates in KC1 plus lysine or dissociates a t low ionic strength in the absence of the ligand. MATERIALS AND METHODS Strains Used and Enzyme PurificationAspartokinase I11 was purified from Escherichia coli K12. I n addition to the strains HfrH grown as previously described [S] two other strains were used which produce larger amounts of enzyme: DO6 is a partially constitutive mutant for aspartokinase I11 isolated in our laboratory (J. C. Patte, unpublished), in which the specific activity of this enzyme is 3 to 5-fold increased when the st.rain is grown in minimal medium. G6 MD3 is a mutant carrying a deletion for the gene coding for aspartic semialdehyde dehydrogenase (asd) [12], kindly provided by M. Schwartz ; when this strain is grown in the presence of 20 pM diaminopimelic acid (plus 0.2 mM m-methionine and 2 mM DL-threonine) the aspartokinase I11 synthesis is 10 to 15-fold derepressed (probably due to lysine limitation).Strain DO6 was grown in fermentor of 200 1 a t ...

Structures of R- and T-stateEscherichia coliAspartokinase III

“…Consistent with reports of lysine-induced AKIII tetramer formation (22,23), association of two dimers is apparent across a crystallographic 2-fold axis of the T-state AKIII (Fig. 1D).…”

“…A change in the oligomeric state of AKIII to a homotetramer on lysine binding has been suggested (18,22,23). Mutational analyses of the C-terminal AKIII region indicate this to be the site for lysine binding (24,25).…”

The initial step in the archaeal aspartate biosynthetic pathway catalyzed by a monofunctional aspartokinase

“…However, native gel electrophoresis studies suggested that the plant enzyme exists as a functional dimer in solution (Mas-Droux et al, 2006) and earlier analytical ultracentrifugation and gel-filtration studies clearly established the dimer as the catalytically active unit of the monofunctional AK in Escherichia coli (ecAKIII; Richaud et al, 1973. Gel-filtration and dynamic light-scattering studies were conducted on mjAK to address the discrepancy in the quaternary structure of AK between that observed in the crystal and that reported in solution.…”

“…The AKs from Escherichia coli include the bifunctional AK-HDH I which assembles into a 360 kDa tetrameric complex (Veron et al, 1985), while the second bifunctional enzyme (AK-HDH II) is found as a dimer in solution (Dautry-Varsat et al, 1977). The monofunctional AK III from E. coli assembles into a smaller dimer of only 96 kDa (Richaud et al, 1973). Additional complexity is evident in the regulation of the AKs, which are mediated by the end products of the pathway.…”

Evolution of biosynthetic pathways: immunological approach.

“…The capacities of the different immunoadsorbents for their homologous antigens were determined as previously reported (18) and are expressed in nanomoles of subunits bound per milliliter of packed immunoadsorbent. The respective molecular weights of the subunits of the enzymes which will be shown to be retained are the following: aspartokinase I-homoserine dehydrogenase I = 86,000 (19); aspartokinase I fragment = 48,000 (10); proteolytic homoserine dehydrogenase I fragment = 55,000 (10); aspartokinase II-homoserine dehydrogenase II = 86,000 (unpublished results); aspartokinase III = 52,000 (20); homoserine kinase = 30,000 (Burr, in preparation).…”