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 (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 | 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 | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required, two Mg2+ ions per enzyme molecule | Thermococcus kodakarensis | |
additional information | LysW contains a Zn2+ | Thermococcus kodakarensis |
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 | - |
ADP + phosphate + an [amino-group carrier protein]-C-terminal-gamma-(L-glutamyl)-L-glutamate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Thermococcus kodakarensis | Q5JFW0 | Pyrococcus kodakaraensis strain KOD1 | - |
Thermococcus kodakarensis ATCC BAA-918 | Q5JFW0 | Pyrococcus kodakaraensis strain KOD1 | - |
Thermococcus kodakarensis JCM 12380 | Q5JFW0 | Pyrococcus kodakaraensis strain KOD1 | - |
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 | 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 Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.016 | - |
pH 8.0, 60°C, recombinant enzyme | Thermococcus kodakarensis |
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 | 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 | 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 Maximum [°C] | Comment | Organism |
---|---|---|---|
60 | - |
assay at | Thermococcus kodakarensis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Thermococcus kodakarensis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Thermococcus kodakarensis |
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 |