scholarly journals Lysine biofortification in rice by modulating feedback inhibition of aspartate kinase and dihydrodipicolinate synthase

2020 ◽  
Author(s):  
Qing‐Qing Yang ◽  
Wai‐Han Yu ◽  
Hong‐Yu Wu ◽  
Chang‐Quan Zhang ◽  
Samuel Sai‐Ming Sun ◽  
...  
Keyword(s):  
Feedback Inhibition ◽  
Aspartate Kinase ◽  
Dihydrodipicolinate Synthase

Cellular Amino Acid Concentrations and Regulation of Aspartate Kinase in the Thermophilic Phototrophic Prokaryote Chloroflexus aurantiacus

1990 ◽  
Vol 45 (1-2) ◽  
pp. 74-78 ◽  
Author(s):  
Jobst-Heinrich Klemme ◽  
Gisela Laakmann-Ditges ◽  
Jutta Mertschuweit
Keyword(s):  
Amino Acid ◽  
Feedback Inhibition ◽  
Gel Filtration ◽  
Break Point ◽  
Test System ◽  
Chloroflexus Aurantiacus ◽  
Aspartate Kinase ◽  
Homoserine Dehydrogenase ◽  
Molecular Weights ◽  
Amino Acid Concentrations

Aspartate kinase (AK , EC 2.7.2.4) from the thermophilic, phototrophic prokaryote, Chloroflexus aurantiacus, was partially purified and separated from homoserine dehydrogenase (HSDH, EC 1.1.1.3). The molecular weights as determined by gel filtration were 130,000 and 46,000, respectively. HSDH had a moderately high thermal stability (50% inactivation at 84 °C) and displayed its activity optimum at 72 °C. By contrast, AK had its activity optimum at 52 °C (with a break-point in the Arrhenius plot at 42 °C) and was much less thermostable (50% inactivation at 67 °C). The Km-values for aspartate and ATP (determined in a pyruvate kinase-coupled test system) were 10.5 and 0.63 mM , respectively. The enzyme was strongly inhibited by L-threonine (Ki = 10 μm) and activated by alanine, isoleucine, valine and methionine. L-Threonine acted as a mixed-type inhibitor in respect to aspartate, and non-competitively in respect to ATP. Contrary to AKs from Rhodospirillaceae, the enzyme from Chloroflexus aurantiacus was not subject to a concerted feedback inhibition by two amino acids of the aspartate family. The regulatory properties of the aspartate kinase are discussed in relation to the cellular amino acid concentrations.

scholarly journals Genetic and biochemical study of threonine-overproducing mutants of Saccharomyces cerevisiae.

1982 ◽  
Vol 2 (7) ◽  
pp. 731-736 ◽  
Author(s):  
M A Delgado ◽  
J Guerrero ◽  
J Conde
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mutant Strain ◽  
Feedback Inhibition ◽  
Biochemical Study ◽  
Aspartate Kinase

Three threonine-overproducing mutants were obtained as prototrophic revertants of a hom3 mutant strain of Saccharomyces cerevisiae. The gene HOM3 codes for aspartokinase (aspartate kinase; EC 2.7.2.4), the first enzyme of the threonine-methionine biosynthetic route, which is subjected to feedback inhibition by threonine. Enzymatic studies indicated that aspartokinase from the revertants has lost the feedback inhibition, resulting in overproduction of threonine. These revertants also bore one or two additional mutations, named tex1-1 and tex2-1, which alone or jointly made possible the excretion of the threonine accumulated. The effect of these two genes on excretion is potentiated by excess inositol in the medium.

scholarly journals Allosteric Signaling in Dihydrodipicolinate Synthase

2014 ◽  
Vol 70 (a1) ◽  
pp. C443-C443
Author(s):  
Cuylar Conly ◽  
Yulia Skovpen ◽  
David Palmer ◽  
David Sanders
Keyword(s):  
Cell Wall ◽  
Active Sites ◽  
Structural Changes ◽  
Feedback Inhibition ◽  
Bacterial Species ◽  
Clear Understanding ◽  
Dihydrodipicolinate Synthase ◽  
Reactive Site ◽  
Ongoing Research ◽  
Natural Inhibitor

Dihydrodipicolinate synthase (DHDPS) is an enzyme found in most bacterial species and regulates the production of cell wall precursors necessary for the life of the organism. Specifically, DHDPS catalyzes the condensation of pyruvate and aspartate-β-semialdehyde (ASA) to produce dihydrodipicolinic acid leading to the synthesis of cell wall precursors lysine and mesodiaminopimelate. DHDPS is regulated through feedback inhibition when lysine binds at a location distinct from the reactive site. The mechanism by which lysine remotely disrupts catalysis is not well understood. A clear understanding of how the natural inhibitor, lysine, binds to DHDPS and what effects this has on the machinery of the enzyme will be invaluable for development of novel antibiotic leads. Analysis of DHDPS crystals with and without inhibitors has revealed structural changes that appear to link the allosteric and active sites. Our ongoing research examines the validity of observed structural changes in the mechanism of allosteric inhibition.

Bacterial Dihydrodipicolinate Synthase and Desensitized Aspartate Kinase: Two Novel Selectable Markers for Plant Transformation

1993 ◽  
Vol 11 (6) ◽  
pp. 715-718 ◽  
Author(s):  
Avihai Perl ◽  
Shmuel Galili ◽  
Orit Shaul ◽  
Inbal Ben-Tzvi ◽  
Gad Galili
Keyword(s):  
Plant Transformation ◽  
Aspartate Kinase ◽  
Dihydrodipicolinate Synthase ◽  
Selectable Markers

scholarly journals Engineering a feedback inhibition-insensitive plant dihydrodipicolinate synthase to increase lysine content in Camelina sativa seeds

2021 ◽  
Author(s):  
Alex Huang ◽  
Cathy Coutu ◽  
Myrtle Harrington ◽  
Kevin Rozwadowski ◽  
Dwayne D. Hegedus
Keyword(s):  
Animal Feed ◽  
Specific Activity ◽  
Higher Plants ◽  
Feedback Inhibition ◽  
Camelina Sativa ◽  
Site Directed Mutagenesis ◽  
Oilseed Crop ◽  
Lysine Content ◽  
Dihydrodipicolinate Synthase ◽  
The Impact

AbstractCamelina sativa (camelina) is emerging as an alternative oilseed crop due to its short growing cycle, low input requirements, adaptability to less favorable growing environments and a seed oil profile suitable for biofuel and industrial applications. Camelina meal and oil are also registered for use in animal and fish feeds; however, like meals derived from most cereals and oilseeds, it is deficient in certain essential amino acids, such as lysine. In higher plants, the reaction catalyzed by dihydrodipicolinate synthase (DHDPS) is the first committed step in the biosynthesis of lysine and is subject to regulation by lysine through feedback inhibition. Here, we report enhancement of lysine content in C. sativa seed via expression of a feedback inhibition-insensitive form of DHDPS from Corynebacterium glutamicums (CgDHDPS). Two genes encoding C. sativa DHDPS were identified and the endogenous enzyme is partially insensitive to lysine inhibition. Site-directed mutagenesis was used to examine the impact of alterations, alone and in combination, present in lysine-desensitized DHDPS isoforms from Arabidopsis thaliana DHDPS (W53R), Nicotiana tabacum (N80I) and Zea mays (E84K) on C. sativa DHDPS lysine sensitivity. When introduced alone, each of the alterations decreased sensitivity to lysine; however, enzyme specific activity was also affected. There was evidence of molecular or structural interplay between residues within the C. sativa DHDPS allosteric site as coupling of the W53R mutation with the N80V mutation decreased lysine sensitivity of the latter, but not to the level with the W53R mutation alone. Furthermore, the activity and lysine sensitivity of the triple mutant (W53R/N80V/E84T) was similar to the W53R mutation alone or the C. glutamicum DHDPS. The most active and most lysine-insensitive C. sativa DHDPS variant (W53R) was not inhibited by free lysine up to 1 mM, comparable to the C. glutamicums enzyme. Seed lysine content increased 13.6 -22.6% in CgDHDPS transgenic lines and 7.6–13.2% in the mCsDHDPS lines. The high lysine-accumulating lines from this work may be used to produce superior quality animal feed with improved essential amino acid profile.

scholarly journals Genetic and biochemical study of threonine-overproducing mutants of Saccharomyces cerevisiae

1982 ◽  
Vol 2 (7) ◽  
pp. 731-736
Author(s):  
M A Delgado ◽  
J Guerrero ◽  
J Conde
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mutant Strain ◽  
Feedback Inhibition ◽  
Biochemical Study ◽  
Aspartate Kinase

Three threonine-overproducing mutants were obtained as prototrophic revertants of a hom3 mutant strain of Saccharomyces cerevisiae. The gene HOM3 codes for aspartokinase (aspartate kinase; EC 2.7.2.4), the first enzyme of the threonine-methionine biosynthetic route, which is subjected to feedback inhibition by threonine. Enzymatic studies indicated that aspartokinase from the revertants has lost the feedback inhibition, resulting in overproduction of threonine. These revertants also bore one or two additional mutations, named tex1-1 and tex2-1, which alone or jointly made possible the excretion of the threonine accumulated. The effect of these two genes on excretion is potentiated by excess inositol in the medium.

Crystal structure and kinetic study of dihydrodipicolinate synthase from Mycobacterium tuberculosis

2008 ◽  
Vol 411 (2) ◽  
pp. 351-360 ◽  
Author(s):  
Georgia Kefala ◽  
Genevieve L. Evans ◽  
Michael D. W. Griffin ◽  
Sean R. A. Devenish ◽  
F. Grant Pearce ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Analytical Ultracentrifugation ◽  
Feedback Inhibition ◽  
Three Dimensional ◽  
Lysine Biosynthesis ◽  
Dimensional Structure ◽  
Dihydrodipicolinate Synthase ◽  
Three Dimensional Structure ◽  
And Function

The three-dimensional structure of the enzyme dihydrodipicolinate synthase (KEGG entry Rv2753c, EC 4.2.1.52) from Mycobacterium tuberculosis (Mtb-DHDPS) was determined and refined at 2.28 Å (1 Å=0.1 nm) resolution. The asymmetric unit of the crystal contains two tetramers, each of which we propose to be the functional enzyme unit. This is supported by analytical ultracentrifugation studies, which show the enzyme to be tetrameric in solution. The structure of each subunit consists of an N-terminal (β/α)8-barrel followed by a C-terminal α-helical domain. The active site comprises residues from two adjacent subunits, across an interface, and is located at the C-terminal side of the (β/α)8-barrel domain. A comparison with the other known DHDPS structures shows that the overall architecture of the active site is largely conserved, albeit the proton relay motif comprising Tyr143, Thr54 and Tyr117 appears to be disrupted. The kinetic parameters of the enzyme are reported: KMASA=0.43±0.02 mM, KMpyruvate=0.17±0.01 mM and Vmax=4.42±0.08 μmol·s−1·mg−1. Interestingly, the Vmax of Mtb-DHDPS is 6-fold higher than the corresponding value for Escherichia coli DHDPS, and the enzyme is insensitive to feedback inhibition by (S)-lysine. This can be explained by the three-dimensional structure, which shows that the (S)-lysine-binding site is not conserved in Mtb-DHDPS, when compared with DHDPS enzymes that are known to be inhibited by (S)-lysine. A selection of metabolites from the aspartate family of amino acids do not inhibit this enzyme. A comprehensive understanding of the structure and function of this important enzyme from the (S)-lysine biosynthesis pathway may provide the key for the design of new antibiotics to combat tuberculosis.

Mechanism of the feedback-inhibition resistance in aspartate kinase of Corynebacterium pekinense: from experiment to MD simulations

RSC Advances ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 30-38
Author(s):  
Xiaoting Liu ◽  
Caijing Han ◽  
Li Fang ◽  
Zhanqing Fan ◽  
Yanan Wang ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Feedback Inhibition ◽  
Md Simulations ◽  
Aspartate Kinase ◽  
High Enzyme ◽  
High Enzyme Activity

Corynebacterium pekinense AK was successfully modified and two mutants A380C and T379N/A380C with high enzyme activity were constructed. The mechanism of their feedback-inhibition resistance was thoroughly studied from experiment to MD simulations.

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