The enzymes from the thermophilic bacterium Thermus thermophilus and the thermophilic archaea Sulfolobus acidocaldarius and Sulfolobus tokodaii protect the amino group of L-2-aminoadipate by conjugation to the carrier protein LysW. This reaction is part of the lysine biosynthesis pathway in these organisms.
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The expected taxonomic range for this enzyme is: Bacteria, Archaea
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ATP + an [amino group carrier protein]-C-terminal-L-glutamate + L-2-aminoadipate = ADP + phosphate + an [amino group carrier protein]-C-terminal-N-(1,4-dicarboxybutyl)-L-glutamine
substrate-recognition mechanism of bifunctional TkLysX/ArgX
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SYSTEMATIC NAME
IUBMB Comments
[amino group carrier protein]-C-terminal-L-glutamate:L-2-aminoadipate ligase (ADP-forming)
The enzymes from the thermophilic bacterium Thermus thermophilus and the thermophilic archaea Sulfolobus acidocaldarius and Sulfolobus tokodaii protect the amino group of L-2-aminoadipate by conjugation to the carrier protein LysW. This reaction is part of the lysine biosynthesis pathway in these organisms.
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
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. Substrate binding structure, overview. Residues Thr196, Asn250, and Ala251 are involved in the recognition of the delta-carboxyl group of AAA
proposal of a mechanism for substrate recognition and its relationship with molecular evolution among LysX family proteins, which have different substrate specificities
the enzyme is part of the lysine biosynthetic pathway in Thermococcus kodakarensis, overview. The gene cluster for lysine biosynthetic genes through AAA produces lysine with a LysW-mediated system. In this cluster, TK0279, TK0278, TK0276, TK0277, TK0275, and TK0274 are expected to be responsible for the conversion process from AAA to lysine through the LysW system as LysW-gamma-AAA synthetase, LysW-gamma-AAA kinase, LysW-gamma-AAA semialdehyde dehydrogenase, LysW-gamma-lysine aminotransferase, and LysW-gamma-lysine carboxypeptidase, respectively. LysW-gamma-AAA thus synthesized is transferred to subsequent biosynthetic enzymes to be converted to LysW-gamma-lysine by phosphorylation, reduction, and amination steps. In the final step, LysW-gamma-lysine is recognized by LysK, a carboxypeptidase, resulting in the release of lysine. The lysine biosynthetic enzymes of Thermococcus kodakarensis convert AAA/Glu to lysine/ornithine
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 has the potential to biosynthesize lysine and ornithine using the carrier protein LysW-mediated system with a single set of bifunctional enzymes
enzyme LysX recognizes alpha-aminoadipate (AAA) as a substrate, structural basis for the bifunctionality of the LysX family protein from Thermococcus kodakarensis. 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
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
purified recombinant nontagged enzyme TkLysX/ArgX in complex with [LysW]-C-terminal-N-(1,5-dicarboxypentan-1-yl)-L-glutamine ((TkLysX/ArgXx02LysW-gamma-AAA)), which corresponds to the structure of the post-reaction state of TkLysX/Arg, hanging drop vapor diffusion method, mixing of 7.5 mg/ml protein and 2.5 mg/ml TkLysW in 10 mM AMP-PNP, 10 mM AAA, or 50 mM glutamate and 10 mM MgSO4 with precipitation solution containing containing 20% v/v 2-methyl-2,4-pentanediol, 6.6% w/v PEG-3350, 0.066 M imidazole, pH 6.5, and 0.132 M ammonium sulfate, 20°C, X-ray diffraction structure determination and analysis at 2.18 A resolution. Molecular replacement. The 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. Good crystals containing TkLysW-gamma-Glu are not obtained
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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