Literature summary for 1.1.1.3 extracted from

Akai, S.; Ikushiro, H.; Sawai, T.; Yano, T.; Kamiya, N.; Miyahara, I.
The crystal structure of homoserine dehydrogenase complexed with L-homoserine and NADPH in a closed form (2019), J. Biochem., 165, 185-195 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene thrA, functional recombinant expression in Escherichia coli strain Rosetta (DE3) pLysS	Thermus thermophilus

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified wild-type homoserine dehydrogenase in apoform, complexed with L-homoserine and NADPH in a closed form, and enzyme mutants K99A and K195A complexed with L-Hse and NADP+, hanging-drop vapour-diffusion method, mixing of 0.002 ml of 5 mg/ml protein in 5 mM Tris-HCl, pH 7.5, and in case of the ligand complex forms 15 mM of each ligand, with 0.002 ml of reservoir solution containing 3.3-4.0 M sodium formate and 50 mM CAPS, pH 10.0, X-ray diffraction structure determination and analysis at 1.83 A, 2.00 A, 1.87 A, and 1.93 A resolution, respectively, molecular replacement method using the TtHSDx02L-Hse structure (PDB ID 5XDF) as the search model	Thermus thermophilus

Crystallization (Comment)

Organism

purified wild-type homoserine dehydrogenase in apoform, complexed with L-homoserine and NADPH in a closed form, and enzyme mutants K99A and K195A complexed with L-Hse and NADP+, hanging-drop vapour-diffusion method, mixing of 0.002 ml of 5 mg/ml protein in 5 mM Tris-HCl, pH 7.5, and in case of the ligand complex forms 15 mM of each ligand, with 0.002 ml of reservoir solution containing 3.3-4.0 M sodium formate and 50 mM CAPS, pH 10.0, X-ray diffraction structure determination and analysis at 1.83 A, 2.00 A, 1.87 A, and 1.93 A resolution, respectively, molecular replacement method using the TtHSDx02L-Hse structure (PDB ID 5XDF) as the search model

Thermus thermophilus

Protein Variants

Protein Variants	Comment	Organism
K195A	site-directed mutagenesis, inactive mutant. In the crystal structure, the positions of Lys195 and L-Hse are significantly retained with those of the wild-type enzyme, enzyme crystal structure analysis	Thermus thermophilus
K99A	site-directed mutagenesis, inactive mutant. In the crystal structure, the productive geometry of the ternary complex is almost preserved with one new water molecule taking over the hydrogen bonds associated with Lys99, mutant enzyme crystal structure analysis	Thermus thermophilus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	Michaelis-Menten steady-state kinetics	Thermus thermophilus
0.11	-	NADP+	pH 10.0, 50°C, recombinant enzyme	Thermus thermophilus
0.2	-	L-homoserine	pH 10.0, 50°C, recombinant enzyme, with NADP+	Thermus thermophilus
0.74	-	NAD+	pH 9.5, 50°C, recombinant enzyme	Thermus thermophilus
1.08	-	L-homoserine	pH 9.5, 50°C, recombinant enzyme, with NAD+	Thermus thermophilus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
L-homoserine + NAD+	Thermus thermophilus	-	L-aspartate 4-semialdehyde + NADH + H+	-	?
L-homoserine + NADP+	Thermus thermophilus	-	L-aspartate 4-semialdehyde + NADPH + H+	-	?

Organism

Organism	UniProt	Comment	Textmining
Thermus thermophilus	Q5SL04	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant enzyme from Escherichia coli strain Rosetta(DE3)pLys by heat treatment at 70°C for 10 min, gel filtration, hydrophobic interaction and anion exchange chromatography, desalting gel filtration, and ultrafiltration, to homogeneity	Thermus thermophilus

Reaction

Reaction	Comment	Organism	Reaction ID
L-homoserine + NAD(P)+ = L-aspartate 4-semialdehyde + NAD(P)H + H+	two possible catalytic mechanisms of homoserine oxidation. (a) Lys99 is the acidx02base catalytic residue. The hydroxy group of L-Hse is activated by protonated Lys195, and a proton from the Od of L-Hse is captured by the deprotonated Lys99. Lys99 exists in the simple proton pathway, in which a proton can go in and out of an active site easily. (b) Lys195 is drown as an acidx02base catalytic residue. L-Hse and Asp191 exist deep inside of the active site and have no interaction to the surface. Thus, the direct proton pathway from Lys195 to the bulk water region cannot be realized. Lys99 and Lys195 of TtHSD are essential for catalysis	Thermus thermophilus	a

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
23	-	purified recombinant enzyme, pH 9.5, 50°C	Thermus thermophilus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
L-homoserine + NAD+	-	Thermus thermophilus	L-aspartate 4-semialdehyde + NADH + H+	-	?
L-homoserine + NADP+	-	Thermus thermophilus	L-aspartate 4-semialdehyde + NADPH + H+	-	?
additional information	substrates binding modes, overview	Thermus thermophilus	?	-	-

Subunits

Subunits	Comment	Organism
?	x * 35492, sequence calculation	Thermus thermophilus
More	enzyme TtHSD folds into a dimer with a noncrystallographic 2fold axis. The subunit comprises three conserved domains of HSDs and a flexible tail at the C-terminus. The nucleotide-binding domain (residues 1-119 and 288-309) assumes an alpha/beta Rossmann fold with five beta-strands and four alpha-helices. The dimerization domain (residues 120-140 and 261-287) comprises two alpha-helices and two beta-strands that interact with the corresponding domain of the other subunit of the dimer to form an alpha/beta structure with the four-stranded beta-sheet. The substrate-binding domain (residues 141-260) comprises four beta-strands and five alpha-helices. The flexible tail at the C-terminus (310-332) extends from the nucleotide-binding domain to the substrate-binding domain	Thermus thermophilus

Synonyms

Synonyms	Comment	Organism
thrA	-	Thermus thermophilus
TTHA0489	-	Thermus thermophilus
TtHSD	-	Thermus thermophilus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
60	70	L-homoserine oxidation	Thermus thermophilus

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
82	-	Tm of wild-type TtHSD	Thermus thermophilus

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
2.12	-	NADP+	pH 10.0, 50°C, recombinant enzyme	Thermus thermophilus
6.66	-	NAD+	pH 9.5, 50°C, recombinant enzyme	Thermus thermophilus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
9.5	-	L-homoserine oxidation	Thermus thermophilus

Cofactor

Cofactor	Comment	Organism
NAD+	-	Thermus thermophilus
NADH	-	Thermus thermophilus
NADP+	-	Thermus thermophilus
NADPH	-	Thermus thermophilus

General Information

General Information	Comment	Organism
evolution	structure-function analysis and comparisons	Thermus thermophilus
additional information	binding of L-Hse and NADPH induces the conformational changes of TtHSD from an open to a closed form: the mobile loop containing Glu180 approaches to fix L-Hse and NADPH, and both Lys99 and Lys195 make hydrogen bonds with the hydroxy group of L-Hse. The ternary complex of TtHSDs in the closed form mimicks a Michaelis complex better than the previously reported open form structures from other species. Lys99 seems to be the acidx02base catalytic residue of HSD. The substrate L-Hse and the nicotinamide-ribose moiety of the cofactor NADPH are bound to a crevice formed at the interface between the substrate and nucleotide binding domains. In contrast, the adenosine group of NADPH is located at the surface of the enzyme. The open-closed conformational change may play an important role in the formation of the enzymex02substrate-cofactor complex and subsequent enzymatic catalysis	Thermus thermophilus

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
6.2	-	NAD+	pH 9.5, 50°C, recombinant enzyme	Thermus thermophilus
9	-	NAD+	pH 9.5, 50°C, recombinant enzyme	Thermus thermophilus
10.6	-	NADP+	pH 10.0, 50°C, recombinant enzyme	Thermus thermophilus
19.27	-	NADP+	pH 10.0, 50°C, recombinant enzyme	Thermus thermophilus