Expression, purification, and biochemical characterization of an NAD+-dependent homoserine dehydrogenase from the symbiotic Polynucleobacter necessarius subsp. necessarius.

Homoserine dehydrogenase (HSD), encoded by the hom gene, is a key enzyme in the aspartate pathway, which reversibly catalyzes the conversion of l-aspartate ß-semialdehyde to l-homoserine (l-Hse), using either NAD(H) or NADP(H) as a coenzyme. In this work, we presented the first characterization of the HSD from the symbiotic Polynucleobacter necessaries subsp. necessarius (PnHSD) produced in Escherichia coli. Sequence analysis showed that PnHSD is an ACT domain-containing monofunctional HSD with 436 amnio acid residues. SDS-PAGE and Western blot demonstrated that PnHSD could be overexpressed in E. coli BL21(DE3) cell as a soluble form by using SUMO fusion technique. It could be purified to apparent homogeneity for biochemical characterization. Size-exclusion chromatography revealed that the purified PnHSD has a native molecular mass of ∼160 kDa, indicating a homotetrameric structure. The oxidation activity of PnHSD was studied in this work. Kinetic analysis revealed that PnHSD displayed an up to 1460-fold preference for NAD+ over NADP+, in contrast to its homologs. The purified PnHSD displayed maximal activity at 35 °C and pH 11. Similar to its NAD+-dependent homolog, neither NaCl and KCl activation nor L-Thr inhibition on the enzymatic activity of PnHSD was observed. These results will contribute to a better understanding of the coenzyme specificity of the HSD family and the aspartate pathway of P. necessarius.

Crystallization and preliminary X-ray diffraction studies of Staphylococcus aureus homoserine dehydrogenase.

Staphylococcus aureus is a Gram-positive nosocomial pathogen. The prevalence of multidrug-resistant S. aureus strains in both hospital and community settings makes it imperative to characterize new drug targets to combat S. aureus infections. In this context, enzymes involved in cell-wall maintenance and essential amino-acid biosynthesis are significant drug targets. Homoserine dehydrogenase (HSD) is an oxidoreductase that is involved in the reversible conversion of L-aspartate semialdehyde to L-homoserine in a dinucleotide cofactor-dependent reduction reaction. HSD is thus a crucial intermediate enzyme linked to the biosynthesis of several essential amino acids such as lysine, methionine, isoleucine and threonine.

Purification and characterization of homoserine dehydrogenase from spinach leaves.

Homoserine dehydrogenase (HSDH) has been purified to homogeneity from spinach leaves using ammonium sulphate fractionation followed by ion exchange chromatography, gel filtration and FPLC techniques. The purified enzyme has a relative molecular mass of 220,000 and subunit molecular mass of 55,000 and probably occurs as a tetramer. The enzyme was found to be sensitive to threonine and also exhibited aspartate kinase (AK) activity, which was also sensitive to threonine suggesting that it is a bifunctional protein. The enzyme protein also gave a positive cross reaction with antibodies raised against purified AK isoenzymes. Both HSDH and AK activities were stimulated by calcium and calmodulin.

Activities and regulation of the enzymes involved in the first and the third steps of the aspartate biosynthetic pathway in Enterococcus faecium.

The enzymes aspartokinase and homoserine dehydrogenase catalyze the reaction at key branching points in the aspartate pathway of amino acid biosynthesis. Enterococcus faecium has been found to contain two distinct aspartokinases and a single homoserine dehydrogenase. Aspartokinase isozymes eluted on gel filtration chromatography at molecular weights greater than 250,000 and about 125,000. The molecular weight of homoserine dehydrogenase was determined to be 220,000. One aspartokinase isozyme was slightly inhibited by meso-diaminopimelic acid. Another aspartokinase was repressed and inhibited by lysine. Although the level of diaminopimelate-sensitive (DAPs) enzyme was not much affected by growth conditions, the activity of lysine-sensitive (Lyss) aspartokinase disappeared rapidly during the stationary phase and was depressed in rich media. The synthesis of homoserine dehydrogenase was controlled by threonine and methionine. Threonine also inhibited the specific activity of this enzyme. The regulatory properties of aspartokinase isozymes and homoserine dehydrogenase from E. faecium are discussed and compared with those from Bacillus subtilis.

Rapid purification and characterization of homoserine dehydrogenase from Saccharomyces cerevisiae.

Homoserine dehydrogenase of Saccharomyces cerevisiae has been rapidly purified to homogeneity by heat and acid treatments, ammonium sulfate fractionation, and chromatography on Matrex Gel Red A and Q-Sepharose columns. The final preparation migrated as a single entity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr of 40,000. The Mr of the native enzyme was 81,000 as determined by gel filtration, suggesting that the enzyme is composed of two identical subunits. This feature was also confirmed by cross-linking analysis using the bifunctional reagent dimethyl suberimidate. Feedback inhibition by L-methionine and L-threonine was observed using the purified enzyme. The enzyme was markedly stabilized against heat treatment at high salt concentrations. Additions of feedback inhibitors or high concentrations of salts failed to cause any dissociation or aggregation of the enzyme subunits unlike enzymes from other sources such as Rhodospirillum rubrum. The enzyme denatured in 3 M guanidine-HCl was refolded by simple dilution with a concomitant restoration of the activity. Cross-linking analysis of the renaturation process suggested that the formation of the dimer is required for activity expression. Amino acid sequence analysis of peptides obtained by digestion of the enzyme protein with Achromobacter lyticus protease I revealed that several amino acid residues are strictly conserved among homoserine dehydrogenases from S. cerevisiae, Escherichia coli, and Bacillus subtilis.

Target of serine inhibition in Escherichia coli.

L-serine has long been known to inhibit growth of Escherichia coli cells cultured in minimal medium supplemented with glucose, lactate, or another carbohydrate as the sole source of carbon. However, the target of serine inhibition was not known. The growth inhibition was released by adding isoleucine, 2-ketobutyric acid, threonine or homoserine, but not by aspartate. Thus the inhibition site must be between aspartate and homoserine in the isoleucine biosynthetic pathway. We found that homoserine dehydrogenase I was strongly inhibited by serine. We isolated serine-resistant mutants, and found that in these mutants homoserine dehydrogenase I was resistant to serine. Thus, we conclude that the target of serine inhibition in Escherichia coli is homoserine dehydrogenase I.

Use of monoclonal antibodies for the purification and characterization of the threonine-sensitive isozyme of maize homoserine dehydrogenase.

Monoclonal antibodies, highly specific for the threonine-sensitive isozyme of maize homoserine dehydrogenase, have been prepared and utilized to purify the enzyme to homogeneity. The results of one- and two-dimensional polyacrylamide gel electrophoresis under denaturing conditions indicate that the enzyme is composed of subunits of identical molecular weight. Apparent microheterogeneity of the subunits was observed during isoelectric focusing, but peptide maps generated by partial cleavage with three different chemical reagents did not reveal any differences among the proteins separated by isoelectric focusing. It is concluded that the subunits of the active dimeric and tetrameric configurations of the maize enzyme are identical or very similar. Evidence is presented which indicates that the enzyme purified by immunoaffinity chromatography retains all of the properties of freshly isolated enzyme, including the ability to undergo several ligand-induced slow transitions among four unique states and complex kinetic responses to physiological substrates. Two monoclonal antibodies are shown to interact differently with the purified enzyme. One, MC-11, reacts with all enzyme molecules, while the other, MC-3, is able to resolve two antigenically distinct subpopulations. These populations are present in approximately equal amounts in etiolated shoots and leaves of light-grown seedlings. However, the results of kinetic and hysteretic studies indicate that they are functionally indistinguishable. The antibodies appear to recognize a structural difference between the enzyme populations which does not result in detectable alterations in their catalytic or regulatory properties.

A hybrid proteolytic fragment of Escherichia coli aspartokinase I-homoserine dehydrogenase I. Structure, inhibition pattern, dissociation properties, and generation of two homodimers.

A hybrid dimeric fragment of Escherichia coli aspartokinase I-homoserine dehydrogenase I (Fazel, A., Müller, K., Le Bras, G., Garel, J.-R., Véron, M., and Cohen, G. N. (1983) Biochemistry 22, 158-165) has been purified and shown to possess both aspartokinase and homoserine dehydrogenase activities and is rather stable in the presence of L-threonine. Its two activities are still inhibited by threonine, but noncooperatively in contrast to the native protein. The aspartokinase activity is found to be more sensitive to threonine than the dehydrogenase activity. In the absence of threonine, the different chains of the hybrid (Mr = 89,000 + 59,000) dissociate first into monomers, this being followed by the pairing of two homologous chains to form two homodimers. In the presence of L-threonine, the two homodimers do not dissociate to re-form the hybrid fragment. The NH2-terminal analysis of different chains of the hybrid shows that the homodimers correspond, respectively, to the dimer of the native protein (Mr = 2 X 89,000) and to a dimer already described (Véron, M., Falcoz-Kelly, F., and Cohen, G. N. (1972) Eur. J. Biochem. 28, 520-527).

Isolation and characterization of two homoserine dehydrogenases from maize suspension cultures.

Homoserine dehydrogenase in unpurified extracts of maize (Zea mays L.) cell suspensions is inhibited 73% by the feedback regulator threonine; the remaining 27% of the total activity is not affected even by high concentrations of threonine. The threonine-resistant and threonine-sensitive homoserine dehydrogenase activities were separated by affinity chromatography on Blue Sepharose columns, and the two distinct homoserine dehydrogenases were purified. The threonine-resistant enzyme is an Mr = 70,000 dimer of two Mr = 38,000 subunits and the threonine-sensitive enzyme is an Mr = 190,000 dimer containing two apparently different subunits with molecular weights of 89,000 and 93,000. The threonine-resistant enzyme exhibits normal Michaelis-Menten kinetics and its activity is not affected by any of the amino acid end products of the aspartate pathway. The threonine-sensitive enzyme exhibits positive cooperative kinetics with respect to NADPH and is inhibited by threonine and stimulated by isoleucine. All attempts to affect interconversion of the two purified enzymes have been unsuccessful. Because the purified enzymes correspond to activities present in crude extracts of various maize tissues, it is concluded that the two types of homoserine dehydrogenase are natural in vivo constituents of maize.

Proteolysis of the bifunctional methionine-repressible aspartokinase II-homoserine dehydrogenase II of Escherichia coli K12. Production of an active homoserine dehydrogenase fragment.

The dimeric bifunctional enzyme aspartokinase II-homoserine dehydrogenase II (Mr = 2 X 88,000) of Escherichia coli K12 can be cleaved into two nonoverlapping fragments by limited proteolysis with subtilisin. These two fragments can be separated under nondenaturing conditions as dimeric species, which indicates that each fragment has retained some of the association areas involved in the conformation of the native protein. The smaller fragment (Mr = 2 X 24,000) is devoid of aspartokinase and homoserine dehydrogenase activity. The larger fragment (Mr = 2 X 37,000) is endowed with full homoserine dehydrogenase activity. These results show that the polypeptide chains of the native enzyme are organized in two different domains, that both domains participate in building up the native dimeric structure, and that one of these domains only is responsible for homoserine dehydrogenase activity. A model of aspartokinase II-homoserine dehydrogenase II is proposed, which accounts for the present results.

Active enzyme gel chromatography. I. Experimental aspects.

Transport properties of active enzyme species can be studied effectively by layering a small band of enzyme-containing sample on a gel chromatographic column previously saturated with substrate. The column is optically scanned at successive time intervals to yield profiles representing the appearance of chromophoric product or disappearnce of chromophoric substrate. These profiles permit determination of the specific activity and rate of transport of the active species. Initial studies on mechanic of the technique establish the feasibility of accurately determining transport properties of active enzyme species chromatographed on gel columns. Illustrative results are presented for L-glutamate dehydrogenase and for homoserine dehydrogenase studied in both forward and reverse reactions. It is shown that the partititon cross sections derived from the rates of motion of catalytic activity are the same as those determined by equilibrium saturation experiments which directly measure the degree of partitioning by the protein. These results establish the validity of the technique for a variety of future studies. Active enzyme gel chromatography appears comparable in precision to the active enzyme sedimentation technique at current stages of development.

Methionine-repressible homoserine dehydrogenase of Serratia marcescens: purification and properties.

Serratia marcescens Sa-3 possesses two homoserine dehydrogenases and neither has any aspartokinase activity unlike the case of Escherichia coli enzymes. The two enzymes have been separated. One of them is active with either NAD+ or NADP+ and has been purified about 180-fold to homogeneity. This enzyme is completely repressed by the presence of 1 mM methionine or homoserine in the growth medium, but its activity is unaffected by any amino acid of the aspartate family either singly or together. In many of its properties (such as pH optimum, Km for substrate and cofactors), it resembles its counterpart in E. coli K12. Potassium ions stabilize the enzyme but are not essential for activity. Its molecular weight is around 155,000 as determined by gel filtration and approximately 76,000 by SDS-polyacrylamide gel electrophoresis. This suggests that the enzyme has two subunits (polypeptide chains) in the molecule: 8 M urea has no effect on enzyme activity. This enzyme represents approximately 30% of the total homoserine dehydrogenase activity of S. marcescens unlike in Salmonella typhimurium and E. coli K12 where it is a minor or a negligible component.