Isolation and Characterization of Dihydrodipicolinate Synthase from Maize

Frisch, David; Gengenbach, B. G.; Tommey, Andrew M.; Sellner, Janita M.; Somers, David A.; Myers, Dorothea E.

doi:10.1104/pp.96.2.444

Cited by 69 publications

(54 citation statements)

References 17 publications

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“…This is not the case, however, as total lysine is approximately 1.5% (2) and the free lysine concentration in the endosperm is maintained at low levels throughout development (2). The free lysine should be maintained at a low concentration because this amino acid is a feedback inhibitor of enzymes involved in early steps of the aspartate pathway (8,10). There is also evidence to suggest that LKR could be under the control of opaque-2, a gene that transactivates the expression of the 22 kD a-zein genes in maize endosperm.…”

Section: Discussionmentioning

confidence: 99%

Partial Purification and Characterization of Lysine-Ketoglutarate Reductase in Normal and Opaque-2 Maize Endosperms

Brochetto-Braga¹,

Leite²,

Arruda³

1992

Plant Physiol.

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Lysine-ketoglutarate reductase catalyzes the first step of lysine catabolism in maize (Zea mays L.) endosperm. The enzyme condenses L-lysine and a-ketoglutarate into saccharopine using NADPH as cofactor. It is endosperm-specific and has a temporal pattern of activity, increasing with the onset of kernel development, reaching a peak 20 to 25 days after pollination, and thereafter decreasing as the kernel approaches maturity. The enzyme was extracted from the developing maize endosperm and partially purified by ammonium-sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Blue-Sepharose CL-6B. The preparation obtained from affinity chromatography was enriched 275-fold and had a specific activity of 411 by the opaque-2 mutation may explain, at least in part, the elevated concentration of lysine found in the opaque-2 endosperm.There is little information on lysine catabolism in higher plants. Most of the available data were obtained in studies on the incorporation and metabolism of radiolabeled precursors by plant tissues. Feeding experiments with ["4C]lysine demonstrated the incorporation of radioactivity into a-amino adipic acid and glutamic acid in wheat (18) and into saccharopine and diaminopimelic acid in maize and barley (16,26). In developing endosperm of maize and barley, radiolabeled lysine is incorporated primarily into glutamic acid and proline (4, 26). These findings indicate that lysine is catabolized in plants via the saccharopine pathway.The first enzymatic evidence for the operation of the saccharopine pathway for lysine catabolism in plants was obtained with the demonstration of LKR3 activity in immature endosperm of maize (3). LKR (EC 1.5.1.8) condenses lysine and a-ketoglutarate into saccharopine using NADPH as cofactor.An understanding of the pathways for lysine biosynthesis and degradation in plants has enormous importance because of the limiting concentration of this essential amino acid in major food sources such as cereals. Valuable information can be obtained by the elucidation of the properties of enzymes involved in the biosynthesis and catabolism of lysine and by the use of mutants in which the activities of the enzymes are altered.Since the discovery of the superior nutritive value of the high lysine maize mutant opaque-2 (15), there have been many studies on the effects of this mutant gene on protein and amino acid metabolism in maize endosperm. The opaque-2 gene is located on the short arm of chromosome 7 and its major effect is the reduction of the maize storage protein zein. This is a complex of polypeptides coded by a multigenic family located on chromosomes 4 and 7 (23). The opaque-2 gene in the homozygous form reduces the zein content of the endosperm by up to 70% (6). The reduction is

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Section: Discussionmentioning

confidence: 99%

Partial Purification and Characterization of Lysine-Ketoglutarate Reductase in Normal and Opaque-2 Maize Endosperms

Brochetto-Braga¹,

Leite²,

Arruda³

1992

Plant Physiol.

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“…DHDPS in higher plants is generally feedback inhibited by low concentrations of (S)-lysine (lmolar range, K i = 11-51 lM) (Mazelis et al 1977;Kumpaisal et al 1987;Frisch et al 1991;Dereppe et al 1992;Ghislain et al 1995), while their counterparts in gram-negative bacteria are only moderately inhibited (K i = 0.4 mM) (Laber et al 1992;Karsten 1997). Interestingly, DHDP synthases from gram-positive bacteria have shown to be insensitive to (S)-lysine (Stahly 1969;Hoganson and Stahly 1975;Cahyanto et al 2006).…”

Section: Introductionmentioning

confidence: 99%

“…Dihydrodipicolinate synthase (DHDPS) (EC 4.2.1.52) is the key enzyme in lysine biosynthesis via the diaminopimelate pathway, which is used by bacteria (Shedlarski and Gilvarg 1970;Cremer et al 1988;Blagova et al 2006;Tsujimoto et al 2006), some phycomycetes and higher plants (Frisch et al 1991;Dereppe et al 1992;Vauterin and Jacobs 1994;Ghislain et al 1995). The enzyme catalyses the condensation of (S)-aspartate-bsemialdehyde ((S)-ASA) and pyruvate to 2,3-dihydrodipicolinic acid (Yugari and Gilvarg 1965) (Fig.…”

Section: Introductionmentioning

confidence: 99%

Characterization of a thermostable dihydrodipicolinate synthase from Thermoanaerobacter tengcongensis

et al. 2008

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Dihydrodipicolinate synthase (DHDPS) catalyses the first reaction of the (S)-lysine biosynthesis pathway in bacteria and plants. The hypothetical gene for dihydrodipicolinate synthase (dapA) of Thermoanaerobacter tengcongensis was found in a cluster containing several genes of the diaminopimelate lysine-synthesis pathway. The dapA gene was cloned in Escherichia coli, DHDPS was subsequently produced and purified to homogeneity. The T. tengcongensis DHDPS was found to be thermostable (T 0.5 = 3 h at 90°C). The specific condensation of pyruvate and (S)-aspartate-b -semialdehyde was catalyzed optimally at 80°C at pH 8.0. Enzyme kinetics were determined at 60°C, as close as possible to in vivo conditions. The established kinetic parameters were in the same range as for example E. coli dihydrodipicolinate synthase. The specific activity of the T. tengcongensis DHDPS was relatively high even at 30°C. Like most dihydrodipicolinate synthases known at present, the DHDPS of T. tengcongensis seems to be a tetramer. A structural model reveals that the active site is well conserved. The binding site of the allosteric inhibitor lysine appears not to be conserved, which agrees with the fact that the DHDPS of T. tengcongensis is not inhibited by lysine under physiological conditions.

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“…Lysine feedback inhibition of DHDPS has been investigated in several plant, Gram-negative and Gram-positive bacterial species to date. Studies involving Daucus carota sativa (Matthews et al, 1979), Pivus sativum (Dereppe et al, 1992), Spinacia aloeracea (Wallsgrove et al, 1980), Triticum aestivium (Kumpaisal et al, 1987), and Zea mays (Frisch et al, 1991) show that DHDPS from plant species are generally strongly inhibited by www.intechopen.com Enzymology of Bacterial Lysine Biosynthesis 229 lysine (IC 50 = 0.01-0.05 mM). In contrast, DHDPS from bacteria are significantly less sensitive to lysine inhibition than their plant counterparts.…”

Section: Regulation Of Dhdps Activitymentioning

confidence: 99%