Literature summary for 1.1.1.3 extracted from

Ogata, K.; Yajima, Y.; Nakamura, S.; Kaneko, R.; Goto, M.; Ohshima, T.; Yoshimune, K.
Inhibition of homoserine dehydrogenase by formation of a cysteine-NAD covalent complex (2018), Sci. Rep., 8, 5749 .

Activating Compound

Activating Compound	Comment	Organism	Structure
additional information	the HSD from the hyperthermophilic archaeon Sulfolobus tokodaii (StHSD) is activated by reductive cleavage of the disulfide bond formed between cysteine residues (Cys304) in the C-terminal regions of the homodimer subunits	Sulfurisphaera tokodaii

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of the enzyme in Escherichia coli strain BL21(DE3)	Sulfurisphaera tokodaii

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified enzyme StHSD complexed with cysteine and NAD+, hanging drop vapour diffusion method, mixing of 0.002 ml of 5 mg/ml protein solution with 0.002 ml of reservoir solution containing 23% w/v PEG 3350, 0.2 M di-ammonium tartrate, 0.001 ml of 20 mM NAD+, and 0.001 ml of 100 mM cysteine, and equilibration against 0.1 ml of reservoir solution, 12°C, X-ray diffraction structure determination and analysis at 2.1 A resolution, molecular replacement using the StHSD structure (PDB ID 4YDR) as an initial phasing model	Sulfurisphaera tokodaii

Crystallization (Comment)

Organism

purified enzyme StHSD complexed with cysteine and NAD+, hanging drop vapour diffusion method, mixing of 0.002 ml of 5 mg/ml protein solution with 0.002 ml of reservoir solution containing 23% w/v PEG 3350, 0.2 M di-ammonium tartrate, 0.001 ml of 20 mM NAD+, and 0.001 ml of 100 mM cysteine, and equilibration against 0.1 ml of reservoir solution, 12°C, X-ray diffraction structure determination and analysis at 2.1 A resolution, molecular replacement using the StHSD structure (PDB ID 4YDR) as an initial phasing model

Sulfurisphaera tokodaii

Inhibitors

Inhibitors	Comment	Organism
L-cysteine	competitive versus L-homoserine, uncompetitive versus cofactors NAD+ and NADP+. 95% inhibition at 10 mM. The feedback inhibition of StHSD by cysteine occurs through the formation of an enzyme-NAD-cysteine complex. Cysteine situates within the homoserine binding site, formation of a covalent bond between cysteine and the nicotinamide ring. Cysteine interacts with six residues (Gly156, Thr157, Tyr183, Glu185, Asp191, and Lys200) in the StHSD active site, binding structure analysis, overview	Sulfurisphaera tokodaii
L-serine	14% inhibition at 10 mM	Sulfurisphaera tokodaii
additional information	tHSD is poorly inhibited by less than 5% by 10 mM L-methionine, L-isoleucine, or L-threonine, all of which are final products in the aspartate pathway, and by L-lysine	Sulfurisphaera tokodaii
NADH	acts as a competitive inhibitor of NAD+, product inhibition, non-competitive inhibition versus L-homoserine	Sulfurisphaera tokodaii

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.33	-	NAD+	pH 8.0, 30°C	Sulfurisphaera tokodaii
1.2	-	NADP+	pH 8.0, 30°C	Sulfurisphaera tokodaii

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
L-homoserine + NAD+	Sulfurisphaera tokodaii	-	L-aspartate 4-semialdehyde + NADH + H+	-	r
L-homoserine + NAD+	Sulfurisphaera tokodaii DSM 16993	-	L-aspartate 4-semialdehyde + NADH + H+	-	r
L-homoserine + NADP+	Sulfurisphaera tokodaii	-	L-aspartate 4-semialdehyde + NADPH + H+	-	r
L-homoserine + NADP+	Sulfurisphaera tokodaii DSM 16993	-	L-aspartate 4-semialdehyde + NADPH + H+	-	r

Organism

Organism	UniProt	Comment	Textmining
Sulfurisphaera tokodaii	F9VNG5	-	-
Sulfurisphaera tokodaii DSM 16993	F9VNG5	-	-

Posttranslational Modification

Posttranslational Modification	Comment	Organism
proteolytic modification	the HSD from the hyperthermophilic archaeon Sulfolobus tokodaii (StHSD) is activated by reductive cleavage of the disulfide bond formed between cysteine residues (Cys304) in the C-terminal regions of the homodimer subunits	Sulfurisphaera tokodaii

Purification (Commentary)

Purification (Comment)	Organism
recombinant enzyme from Escherichia coli strain BL21(DE3) by anion exchange chromatography, dialysis, and ultrafiltration	Sulfurisphaera tokodaii

Reaction

Reaction	Comment	Organism	Reaction ID
L-homoserine + NAD(P)+ = L-aspartate 4-semialdehyde + NAD(P)H + H+	L-homoserine oxidation by StHSD proceeds through a sequentially ordered mechanism in which NAD+ binds to the free form of the enzyme, after which L-homoserine binds to the enzyme-NAD+ complex	Sulfurisphaera tokodaii	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
L-homoserine + NAD+	-	Sulfurisphaera tokodaii	L-aspartate 4-semialdehyde + NADH + H+	-	r
L-homoserine + NAD+	-	Sulfurisphaera tokodaii DSM 16993	L-aspartate 4-semialdehyde + NADH + H+	-	r
L-homoserine + NADP+	-	Sulfurisphaera tokodaii	L-aspartate 4-semialdehyde + NADPH + H+	-	r
L-homoserine + NADP+	-	Sulfurisphaera tokodaii DSM 16993	L-aspartate 4-semialdehyde + NADPH + H+	-	r

Subunits

Subunits	Comment	Organism
dimer	StHSD is composed of a nucleotide-binding region (residues 1-130 and 285-304), a dimerization region (residues 131-145 and 256-284), and a catalytic region (residues 146-255). Presence of a disulfide bond formed between two cysteine residues (position 304) in the C-terminal regions of the two subunits	Sulfurisphaera tokodaii

Synonyms

Synonyms	Comment	Organism
hom	-	Sulfurisphaera tokodaii
HSD	-	Sulfurisphaera tokodaii
StHSD	-	Sulfurisphaera tokodaii

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Sulfurisphaera tokodaii

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Sulfurisphaera tokodaii

Cofactor

Cofactor	Comment	Organism
NAD+	binding structure analysis, overview	Sulfurisphaera tokodaii
NADH	-	Sulfurisphaera tokodaii
NADP+	StHSD also exhibits activity with NADP+, but with much lower efficiency compared to NAD+	Sulfurisphaera tokodaii
NADPH	-	Sulfurisphaera tokodaii

IC50 Value

IC50 Value	IC50 Value Maximum	Comment	Organism	Inhibitor
0.011	-	versus L-homoserine, pH 8.0, 30°C	Sulfurisphaera tokodaii	L-cysteine
0.55	-	versus NAD+, pH 8.0, 30°C	Sulfurisphaera tokodaii	L-cysteine
1.2	-	versus NADP+, pH 8.0, 30°C	Sulfurisphaera tokodaii	L-cysteine

General Information

General Information	Comment	Organism
physiological function	homoserine dehydrogenase (HSD) is an important regulatory enzyme in the aspartate pathway, which mediates synthesis of methionine, threonine and isoleucine from aspartate	Sulfurisphaera tokodaii