Homoserine kinase inhibition

As mentioned earlier, L-threonine production can be enhanced by engineering the export or uptake system. An efficient L-threonine producer strain of E. coli KY10935, which was derived from the wild-type strain by multiple rounds of random mutation and selection, was able to produce 100 g L-1 L-threonine after 77 h cultivation [53]. In this strain, the two key enzymes in the L-threonine biosynthesis (homoserine dehydrogenase and homoserine kinase) were identified to be still inhibited by much lower intracellular concentrations of L-threonine than... 

Baum, H.J. Madison, J.T. Thompson, J.E Feedback inhibition of homoserine kinase from radish leaves. Phytochemistry, 22, 2409-2412 (1983)... 

Shames, S.L. Wedler, RC. Homoserine kinase of Escherichia coli kinetic mechanism and inhibition by L-aspartate semialdehyde. Arch. Biochem. Biophys., 235, 359-370 (1984)... 

Finkelnburg, B. Klemme, J.H. Homoserine kinase from phototrophic bacterium RhodospirUlum rubrum is not sensitive to feedback inhibition by l-Thr. FEMS Microbiol. Lett., 48, 93-96 (1987)... 

Theze, J. Kleidman, L. Saint Girons, L Homoserine kinase from Escherichia coli K-12 properties, inhibition by L-threonine, and regulation of biosynthesis. J. Bacteriol., 118, 577-581 (1974)... 

Ramos, C. Delgado, M.A. Calderon, I.L. Inhibition by different amino acids of the aspartate kinase and the homoserine kinase of the yeast Sac-charomyces cerevisiae. FEBS Lett., 278, 123-126 (1991)... 

In addition to the major elfectors (AdoMet, threonine, and lysine), cysteine and isoleucine may participate in the control of methionine biosynthesis, at least in some plants. Both isoleucine and cysteine would be expected to accumulate as a result of the diversion of O-phosphohomoserine toward threonine. Isoleucine is a potent competitive inhibitor of the homoserine kinase of pea seedlings (Thoenef aL, 1978), but not that of barley seedlings (Aarnes, 1976). Cysteine inhibits homoserine dehydrogenase (see Bryan, this volume. Chapter 11) and can inhibit the stimulation by AdoMet of some (Madison and Thompson, 1976) but not all (Aarnes, 1978 Thoen et al., 1978) preparations of threonine synthase. Any regulatory effect of cysteine may, however, be of short duration since the combined mechanisms described in Section II,D for regulation of cysteine biosynthesis would be expected to restore the normal concentration of this amino acid. Details of the control of methionine biosynthesis by the major effectors AdoMet, threonine, and lysine are presented below. 

The inhibition of E. coli homoserine kinase is complicated. It is inhibited by the substrates, L-homoserine at a concentration above 1 mM and ATP above 3 mM in a hypothetical preferred order manner, by L-threonine in a competitive manner, and by L-lysine in a non-competitive manner (Chassagnole et al. 2001). 

Homoserine kinase hsis been enriched about fifteen- to twenty-fivefold from brewer s yeast extracts (106,107). ATP is essential for the reaction, as would be smticipated, and evidence was obtained of activation by Mg++, Mn , or Zn++. Enzyme activity was found to be inhibited by ethylene-diaminetetraacetate (EDTA) and p-chloromercuribenzoate (106). 

H139L <4> (<4>, mutant enzyme with diminished kinase activity and ATPase activity 150fold greater than that of the wild-type enzyme [12]) [12] H202L <4> (<4>, Km-value for L-homoserine and ATP remain unchanged, the K -value for substrate inhibition by L-homoserine increases about 8fold, the turnover-number decreases by 50%, unlike the wild-type enzyme the L-homoserine ethyl, isopropyl, and n-propyl esters show substrate inhibition [12]) [12]... 

Lysine plus threonine severely inhibits growth of maize in a synergistic manner. Growth inhibition could result from combined effects of lysine on aspartate kinase and threonine on homoserine dehydrogenase, resulting in starvation for methionine. Growth inhibition by lysine -i- threonine can be overcome by supplying methionine. Bryan [1980) Miflin [1977). 

The cells could produce even more lysine if the kinase were to remain active but insensitive to a high lysine concentration. Mutants of the homoserine auxotrophs of C. glutamicum were isolated with this property by growing the organism in the presence of toxic isosteres (close structural analogues) of lysine (e.g. S-(2-aminoethyl)-L-cysteine). One way in which the cells can become resistant to the toxic isostere is to overproduce lysine. This is likely to occur in cells where the mutation alters aspartyl kinase in such a way as to make it insensitive to inhibition by lysine, while allowing it to retain its full catalytic activity. 

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