Literature summary for 6.3.2.43 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 .

Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli	Thermococcus kodakarensis
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
in presence of carrier protein LysW and AMP-PNP, MgSO4, and L-2-aminoadipate or glutamate, to 2.18 A resolution	Thermococcus kodakarensis
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	Thermococcus kodakarensis

Crystallization (Comment)

Organism

in presence of carrier protein LysW and AMP-PNP, MgSO4, and L-2-aminoadipate or glutamate, to 2.18 A resolution

Thermococcus kodakarensis

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

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
Y175I/I185Y/N250G/S251F	mutant exhibits an apparent substrate preference for glutamate	Thermococcus kodakarensis

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required, nine Mg2+ ions per dimeric enzyme tetramer	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 + an [amino group carrier protein]-C-terminal-L-glutamate + L-2-aminoadipate	Thermococcus kodakarensis	-	ADP + phosphate + an [amino group carrier protein]-C-terminal-N-(1,5-dicarboxypentan-1-yl)-L-glutamine	-	?

Organism

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

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 + 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	Thermococcus kodakarensis	a

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
0.016	-	substrate L-glutamate, pH not specified in the publication, temperature not specified in the publication	Thermococcus kodakarensis
0.021	-	substrate L-2-aminoadipate, pH not specified in the publication, temperature not specified in the publication	Thermococcus kodakarensis
0.021	-	pH 8.0, 60°C, recombinant enzyme	Thermococcus kodakarensis

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + an [amino group carrier protein]-C-terminal-L-glutamate + L-2-aminoadipate	-	Thermococcus kodakarensis	ADP + phosphate + an [amino group carrier protein]-C-terminal-N-(1,5-dicarboxypentan-1-yl)-L-glutamine	-	?
ATP + an [amino group carrier protein]-C-terminal-L-glutamate + L-2-aminoadipate	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	Thermococcus kodakarensis	ADP + phosphate + an [amino group carrier protein]-C-terminal-N-(1,5-dicarboxypentan-1-yl)-L-glutamine	-	?
ATP + [lysine-biosynthesis-protein LysW]-C-terminal-L-glutamate + L-2-aminoadipate	-	Thermococcus kodakarensis	ADP + phosphate + [lysine-biosynthesis-protein LysW]-C-terminal-gamma-(L-2-aminoadip-2-yl)-L-glutamate	-	?
ATP + [lysine-biosynthesis-protein LysW]-C-terminal-L-glutamate + L-2-aminoadipate	-	Thermococcus kodakarensis JCM 12380	ADP + phosphate + [lysine-biosynthesis-protein LysW]-C-terminal-gamma-(L-2-aminoadip-2-yl)-L-glutamate	-	?
ATP + [lysine-biosynthesis-protein LysW]-C-terminal-L-glutamate + L-2-aminoadipate	-	Thermococcus kodakarensis ATCC BAA-918	ADP + phosphate + [lysine-biosynthesis-protein LysW]-C-terminal-gamma-(L-2-aminoadip-2-yl)-L-glutamate	-	?
ATP + [lysine-biosynthesis-protein LysW]-C-terminal-L-glutamate + L-glutamate	-	Thermococcus kodakarensis	ADP + phosphate + [lysine-biosynthesis-protein LysW]-C-terminal-gamma-(L-glutam-2-yl)-L-glutamate	-	?
ATP + [LysW mutant E42R]-C-terminal-L-glutamate + L-2-aminoadipate	-	Thermococcus kodakarensis	ADP + phosphate + [LysW mutant E42R]-C-terminal-N-(1,5-dicarboxypentan-1-yl)-L-glutamine	-	?
ATP + [LysW]-C-terminal-L-glutamate + L-2-aminoadipate	-	Thermococcus kodakarensis	ADP + phosphate + [LysW]-C-terminal-N-(1,4-dicarboxybutan-1-yl)-L-glutamine	-	?
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	?	-	-
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 JCM 12380	?	-	-
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 ATCC BAA-918	?	-	-

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
LysX	-	Thermococcus kodakarensis
More	cf. EC 6.3.2.60	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
metabolism	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	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	Thermococcus kodakarensis has the potential to biosynthesize lysine and ornithine using the carrier protein LysW-mediated system with a single set of bifunctional enzymes	Thermococcus kodakarensis
physiological function	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	Thermococcus kodakarensis