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Literature summary for 6.3.2.60 extracted from

  • Yoshida, A.; Tomita, T.; Atomi, H.; Kuzuyama, T.; Nishiyama, M.
    Lysine biosynthesis of Thermococcus kodakarensis with the capacity to function as an ornithine biosynthetic system (2016), J. Biol. Chem., 291, 21630-21643 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
gene TK0278, recombinant expression of C-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21-CodonPlus (DE3)-RIL, recombinant expression of nontagged enzyme in Escherichia coli strain BL21(DE3) Thermococcus kodakarensis

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant nontagged enzyme in complex with LysW and L-glutamate, good crystals containing TkLysW-gamma-Glu are not obtained Thermococcus kodakarensis

Protein Variants

Protein Variants Comment Organism
I185Y site-directed mutagenesis, the mutant exhibits an apparent substrate preference for glutamate Thermococcus kodakarensis
additional information the N250G/A251F mutant is not produced as a soluble protein Thermococcus kodakarensis
N250G site-directed mutagenesis, the mutant exhibits an apparent substrate preference for glutamate Thermococcus kodakarensis
S251F site-directed mutagenesis, the mutant exhibits an apparent substrate preference for glutamate Thermococcus kodakarensis
Y175I site-directed mutagenesis, the mutant exhibits an apparent substrate preference for glutamate Thermococcus kodakarensis

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required, two Mg2+ ions per enzyme molecule Thermococcus kodakarensis
additional information LysW contains a Zn2+ Thermococcus kodakarensis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + L-glutamate + an [amino-group carrier protein]-C-terminal-L-glutamate Thermococcus kodakarensis
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ADP + phosphate + an [amino-group carrier protein]-C-terminal-gamma-(L-glutamyl)-L-glutamate
-
?

Organism

Organism UniProt Comment Textmining
Thermococcus kodakarensis Q5JFW0 Pyrococcus kodakaraensis strain KOD1
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Thermococcus kodakarensis ATCC BAA-918 Q5JFW0 Pyrococcus kodakaraensis strain KOD1
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Thermococcus kodakarensis JCM 12380 Q5JFW0 Pyrococcus kodakaraensis strain KOD1
-

Purification (Commentary)

Purification (Comment) Organism
recombinant C-terminally His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21-CodonPlus (DE3)-RIL by heat treatment at 80°C for 20 min and nickel affinity chromatography. Recombinant nontagged enzyme from Escherichia coli strain BL21(DE3) by heat treatment at 80°C for 20 min, ammonium sulfate fractionation, hydrophobic interaction chromatography, ultrafiltration, and gel filtration Thermococcus kodakarensis

Reaction

Reaction Comment Organism Reaction ID
ATP + L-glutamate + an [amino-group carrier protein]-C-terminal-L-glutamate = ADP + phosphate + an [amino-group carrier protein]-C-terminal-gamma-(L-glutamyl)-L-glutamate substrate-recognition mechanism of bifunctional TkLysX/ArgX Thermococcus kodakarensis

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.016
-
pH 8.0, 60°C, recombinant enzyme Thermococcus kodakarensis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + L-glutamate + an [amino-group carrier protein mutant E42R]-C-terminal-L-glutamate
-
Thermococcus kodakarensis ADP + phosphate + an [amino-group carrier protein mutant E42R]-C-terminal-gamma-(L-glutamyl)-L-glutamate
-
?
ATP + L-glutamate + an [amino-group carrier protein]-C-terminal-L-glutamate
-
Thermococcus kodakarensis ADP + phosphate + an [amino-group carrier protein]-C-terminal-gamma-(L-glutamyl)-L-glutamate
-
?
ATP + L-glutamate + an [amino-group carrier protein]-C-terminal-L-glutamate the amino-group carrier protein is LysW. Determination of the crystal structure of the LysX family protein from Thermococcus kodakarensis, which catalyzes the conjugation of LysW with either alpha-aminoadipate (AAA) or glutamate, in a complex with LysW-gamma-AAA. Residue Tyr175 in TkLysX/ArgX plays a critical role in the recognition of glutamate Thermococcus kodakarensis ADP + phosphate + an [amino-group carrier protein]-C-terminal-gamma-(L-glutamyl)-L-glutamate
-
?
additional information purified recombinant TK0278 recognizes alpha-aminoadipate (AAA) and glutamate as substrates and ligated both compounds with TK0279, an amino group carrier protein LysW in this microorganism, in an ATP-dependent manner Thermococcus kodakarensis ?
-
-

Subunits

Subunits Comment Organism
tetramer the crystal asymmetric unit contains two TkLysX/ArgX tetramers, two intact TkLysW monomers each with one zinc atom, four partial TkLysW, eight ADP molecules, five phosphate ions, two sulfate ions, nine magnesium atoms, one free AAA molecule, and 628 water molecules, binding structure, overview Thermococcus kodakarensis

Synonyms

Synonyms Comment Organism
ArgX
-
Thermococcus kodakarensis
More cf. EC 6.3.2.43 Thermococcus kodakarensis
RimK-related lysine biosynthesis protein UniProt Thermococcus kodakarensis
Tk0278
-
Thermococcus kodakarensis
TkLysX/ArgX
-
Thermococcus kodakarensis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
60
-
assay at Thermococcus kodakarensis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Thermococcus kodakarensis

Cofactor

Cofactor Comment Organism Structure
ATP
-
Thermococcus kodakarensis

General Information

General Information Comment Organism
evolution proposal of a mechanism for substrate recognition and its relationship with molecular evolution among LysX family proteins, which have different substrate specificities Thermococcus kodakarensis
malfunction enzyme mutants Y175I, I185Y, N250G, and S251F exhibit an apparent substrate preference for glutamate, suggesting that Tyr at 185 compensates for the function of Tyr175 in the recognition of the gamma-carboxyl group of glutamate Thermococcus kodakarensis
metabolism the enzyme is part of the lysine biosynthetic pathway in Thermococcus kodakarensis, overview. The lysine biosynthetic enzymes of Thermococcus kodakarensis convert AAA/Glu to lysine/ornithine Thermococcus kodakarensis
additional information the modification by TK0278 and successive phosphorylation by TK0276 occurr at the C terminus of TkLysW. Residue Tyr175 in TkLysX/ArgX plays a critical role in the recognition of glutamate. Residues Thr196, Asn250, and Ala251 are involved in the recognition of the delta-carboxyl group of AAA. The Tyr residue at strand beta10, the Tyr residue at strand beta11, and the Thr residue at the large loop as the additional key residues that define substrate specificity in LysX family proteins Thermococcus kodakarensis
physiological function purified recombinant TK0278 recognizes alpha-aminoadipate (AAA) and glutamate as substrates and ligated both compounds with TK0279, an amino group carrier protein LysW in this microorganism, in an ATP-dependent manner. Proposal of a mechanism for substrate recognition Thermococcus kodakarensis