Literature summary for 1.1.1.79 extracted from

Hoover, G.J.; Jorgensen, R.; Rochon, A.; Bajwa, V.S.; Merrill, A.R.; Shelp, B.J.
Identification of catalytically important amino acid residues for enzymatic reduction of glyoxylate in plants (2013), Biochim. Biophys. Acta, 1834, 2663-2671.

Cloned(Commentary)

Cloned (Comment)	Organism
gene GLYR1, sequence comparisons of GLYR genes and HPR genes	Arabidopsis thaliana
gene GLYR1, sequence comparisons of GLYR genes and HPR genes, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 pLysS	Arabidopsis thaliana
gene GLYR2, sequence comparisons of GLYR genes and HPR genes, recombinant expression of a His6-tagged truncated AtGLYR2 cDNA sequence, lacking the N-terminal 58 amino acids, in Escherichia coli strain BL21 pLysS	Arabidopsis thaliana

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified apo-enzyme, sitting drop vapor diffusion method, mixing of 0.002 ml of protein solution with 0.002 ml of reservoir solution containing 0.2 M calcium acetate hydrate, 20% PEG 3350, pH 6.5, 20°C, 6 weeks, X-ray diffraction structure determination and analysis at 2.1 A resolution, molecular replacement using a previously unrecognized member of the beta-HAD family, cytokine-like nuclear factor, structure	Arabidopsis thaliana

Crystallization (Comment)

Organism

purified apo-enzyme, sitting drop vapor diffusion method, mixing of 0.002 ml of protein solution with 0.002 ml of reservoir solution containing 0.2 M calcium acetate hydrate, 20% PEG 3350, pH 6.5, 20°C, 6 weeks, X-ray diffraction structure determination and analysis at 2.1 A resolution, molecular replacement using a previously unrecognized member of the beta-HAD family, cytokine-like nuclear factor, structure

Arabidopsis thaliana

Protein Variants

Protein Variants	Comment	Organism
D239A	site-directed mutagenesis	Arabidopsis thaliana
F231A	site-directed mutagenesis	Arabidopsis thaliana
K170A	site-directed mutagenesis, catalytically inactive mutant	Arabidopsis thaliana
K170E	site-directed mutagenesis, the mutant shows highly reduced kcat for glyoxylate compared to the wild-type	Arabidopsis thaliana
K170H	site-directed mutagenesis, the mutant shows highly reduced kcat for glyoxylate compared to the wild-type	Arabidopsis thaliana
K170R	site-directed mutagenesis, the mutant shows highly reduced kcat for glyoxylate compared to the wild-type	Arabidopsis thaliana
N174A	site-directed mutagenesis	Arabidopsis thaliana
S121A	site-directed mutagenesis	Arabidopsis thaliana
T95A	site-directed mutagenesis	Arabidopsis thaliana

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	Michaelis-Menten kinetics	Arabidopsis thaliana
0.0009	-	NADPH	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant N174A	Arabidopsis thaliana
0.0018	-	NADPH	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant S121A	Arabidopsis thaliana
0.002	-	NADPH	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant F231A	Arabidopsis thaliana
0.0022	-	NADPH	pH 7.8, temperature not specified in the publication, value determined with the use of a double beam spectrophotometer	Arabidopsis thaliana
0.0027	-	NADPH	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant D239A	Arabidopsis thaliana
0.0034	-	NADPH	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant wild-type enzyme	Arabidopsis thaliana
0.0045	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a double beam spectrophotometer	Arabidopsis thaliana
0.016	-	glyoxylate	pH 7.8, temperature not specified in the publication, recombinant truncated enzyme	Arabidopsis thaliana
0.018	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant wild-type enzyme	Arabidopsis thaliana
0.033	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant K170E	Arabidopsis thaliana
0.061	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant K170R	Arabidopsis thaliana
0.0648	-	NADPH	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant T95A	Arabidopsis thaliana
0.088	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant N174A	Arabidopsis thaliana
0.181	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant S121A	Arabidopsis thaliana
3	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant D239A	Arabidopsis thaliana
4.6	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant T95A	Arabidopsis thaliana
12.4	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant F231A	Arabidopsis thaliana

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
chloroplast	-	Arabidopsis thaliana	9507	-
cytosol	-	Arabidopsis thaliana	5829	-

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
glycolate + NADP+	Arabidopsis thaliana	-	glyoxylate + NADPH + H+	-	r
additional information	Arabidopsis thaliana	HPR3 prefers NADPH over NADH and converts glyoxylate to glycolate, the purified recombinant HPR3 shows similar activity with hydroxypyruvate and glyoxylate	?	-	?
additional information	Arabidopsis thaliana	the recombinant AtGLYR1 prefers NADPH over NADH and converts glyoxylate to glycolate, AtGLYR1 has negligible hydroxypyruvate-dependent activity. Isozyme AtGLYR1 also converts succinic semialdehyde to gamma-hydroxybutyrate, albeit with much lower catalytic efficiency than for glyoxylate	?	-	?
additional information	Arabidopsis thaliana	the recombinant AtGLYR2 prefers NADPH over NADH and converts glyoxylate to glycolate, AtGLYR2 has negligible hydroxypyruvate-dependent activity. Isozyme AtGLYR2 also converts succinic semialdehyde to gamma-hydroxybutyrate, albeit with much lower catalytic efficiency than for glyoxylate	?	-	?
additional information	Arabidopsis thaliana	the recombinant AtHPR2 prefers NADPH over NADH but utilizes hydroxypyruvate and glyoxylate similarly	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Arabidopsis thaliana	A0A178WMD4	-	-
Arabidopsis thaliana	F4I907	-	-
Arabidopsis thaliana	Q9CA90	-	-
Arabidopsis thaliana	Q9LSV0	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-tagged truncated mutant enzyme from Escherichia coli strain BL21 pLysS by precipitation with 10% PEG 8000, and nickel affinity chromatography	Arabidopsis thaliana
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 pLysS by precipitation with 10% PEG 8000, and nickel affinity chromatography	Arabidopsis thaliana

Reaction

Reaction	Comment	Organism	Reaction ID
glycolate + NADP+ = glyoxylate + NADPH + H+	the enzyme performs an acid/base catalytic mechanism involving Lys170 as the general acid and a conserved active-site water molecule	Arabidopsis thaliana	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
glycolate + NADP+	-	Arabidopsis thaliana	glyoxylate + NADPH + H+	-	r
glyoxylate + NADPH + H+	-	Arabidopsis thaliana	glycolate + NADP+	-	r
additional information	HPR3 prefers NADPH over NADH and converts glyoxylate to glycolate, the purified recombinant HPR3 shows similar activity with hydroxypyruvate and glyoxylate	Arabidopsis thaliana	?	-	?
additional information	the recombinant AtGLYR1 prefers NADPH over NADH and converts glyoxylate to glycolate, AtGLYR1 has negligible hydroxypyruvate-dependent activity. Isozyme AtGLYR1 also converts succinic semialdehyde to gamma-hydroxybutyrate, albeit with much lower catalytic efficiency than for glyoxylate	Arabidopsis thaliana	?	-	?
additional information	the recombinant AtGLYR2 prefers NADPH over NADH and converts glyoxylate to glycolate, AtGLYR2 has negligible hydroxypyruvate-dependent activity. Isozyme AtGLYR2 also converts succinic semialdehyde to gamma-hydroxybutyrate, albeit with much lower catalytic efficiency than for glyoxylate	Arabidopsis thaliana	?	-	?
additional information	the recombinant AtHPR2 prefers NADPH over NADH but utilizes hydroxypyruvate and glyoxylate similarly	Arabidopsis thaliana	?	-	?

Subunits

Subunits	Comment	Organism
More	domain I, with the dinucleotide binding region, comprises residues 1-165 in the N-terminus. This typical Rossmann fold domain contains two alpha/beta units: a six-stranded parallel beta-sheet (beta1-beta6a) covered by four helices (alpha1-alpha5) and followed by a mixed three-stranded beta-sheet (beta6b-beta8) covered by two helices (alpha6 and alpha7). Domain II (residues 195-287) consists of only helices (alpha8-alpha13) from the C-terminal segment of the protein. The two domains are connected by a long alpha-helix, alpha8 (residues 166-194). Enzyme domain structure analysis, overview	Arabidopsis thaliana

Synonyms

Synonyms	Comment	Organism
At3g25530	-	Arabidopsis thaliana
AtGLYR1	-	Arabidopsis thaliana
AtGLYR2	-	Arabidopsis thaliana
glycerate dehydrogenase	-	Arabidopsis thaliana
GLYR1	-	Arabidopsis thaliana
GLYR2	-	Arabidopsis thaliana
HPR2	-	Arabidopsis thaliana
HPR3	-	Arabidopsis thaliana
More	cf. EC 1.1.1.26	Arabidopsis thaliana

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.0052	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant K170E	Arabidopsis thaliana
0.051	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant K170R	Arabidopsis thaliana
6.06	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant N174A	Arabidopsis thaliana
9.09	-	NADPH	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant N174A	Arabidopsis thaliana
9.56	-	NADPH	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant F231A	Arabidopsis thaliana
11	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant F231A	Arabidopsis thaliana
22	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant D239A	Arabidopsis thaliana
25.4	-	NADPH	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant D239A	Arabidopsis thaliana
51.1	-	NADPH	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant T95A	Arabidopsis thaliana
54.6	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant wild-type enzyme	Arabidopsis thaliana
67.8	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant T95A	Arabidopsis thaliana
84.1	-	NADPH	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant wild-type enzyme	Arabidopsis thaliana
86.4	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant S121A	Arabidopsis thaliana
93.7	-	NADPH	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant S121A	Arabidopsis thaliana
3407	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant wild-type enzyme	Arabidopsis thaliana

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.8	-	assay at	Arabidopsis thaliana

Cofactor

Cofactor	Comment	Organism
additional information	recombinant AtGLYR1 prefers NADPH over NADH	Arabidopsis thaliana
additional information	recombinant AtGLYR2 prefers NADPH over NADH	Arabidopsis thaliana
additional information	recombinant HPR3 prefers NADPH over NADH	Arabidopsis thaliana
additional information	the recombinant AtHPR2 prefers NADPH over NADH	Arabidopsis thaliana
NADP+	-	Arabidopsis thaliana
NADPH	-	Arabidopsis thaliana

General Information

General Information	Comment	Organism
evolution	the enzyme belongs to the beta-HAD (beta-hydroxyacid dehydrogenase) protein family	Arabidopsis thaliana
evolution	the enzyme belongs to the beta-HAD (beta-hydroxyacid dehydrogenase) protein family. AtHPR2 and AtHPR3 are 45% identical to each other at the amino acid level, but only 19-25% identical to AtHPR1, the NADH-dependent form, and 8-9% identical to the AtGLYRs. None of the AtHPRs contains the active-site residues conserved in AtGLYR1 and AtGLYR2, indicating that the sites responsible for reducing glyoxylate differ greatly between the AtGLYRs and AtHPRs	Arabidopsis thaliana
evolution	the primary sequence of cytosolic AtGLYR1 reveals several sequence elements that are consistent with the beta-HAD (beta-hydroxyacid dehydrogenase) protein family, sequence alignment of AtGLYR1 and beta-HAD family members, overview. AtHPR2 and AtHPR3 are 45% identical to each other at the amino acid level, but only 19-25% identical to AtHPR1, the NADH-dependent form, and 8-9% identical to the AtGLYRs. None of the AtHPRs contains the active-site residues conserved in AtGLYR1 and AtGLYR2, indicating that the sites responsible for reducing glyoxylate differ greatly between the AtGLYRs and AtHPRs	Arabidopsis thaliana
evolution	the primary sequence of plastidial AtGLYR2 reveals several sequence elements that are consistent with the beta-HAD (beta-hydroxyacid dehydrogenase) protein family, sequence alignment of AtGLYR2 and beta-HAD family members, overview. AtHPR2 and AtHPR3 are 45% identical to each other at the amino acid level, but only 19-25% identical to AtHPR1, the NADH-dependent form, and 8-9% identical to the AtGLYRs. None of the AtHPRs contains the active-site residues conserved in AtGLYR1 and AtGLYR2, indicating that the sites responsible for reducing glyoxylate differ greatly between the AtGLYRs and AtHPRs	Arabidopsis thaliana
additional information	identification of catalytically important amino acid residues for enzymatic reduction of glyoxylate in plants by bifunctional enzyme glyoxylate/succinic semialdehyde reductase 1, that converts both glyoxylate and succinic semialdehyde into their corresponding hydroxyacid equivalents. Residue Lys170 is essential for catalysis, Phe231, Asp239, Ser121 and Thr95 are more important in substrate binding than in catalysis, and Asn174 is more important in catalysis. Residues Thr95, Phe231 and Asp239 serve a more important role in substrate orientation and docking than in catalysis	Arabidopsis thaliana

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.19	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant K170E	Arabidopsis thaliana
0.86	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant K170R	Arabidopsis thaliana
0.87	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant F231A	Arabidopsis thaliana
7.45	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant D239A	Arabidopsis thaliana
14.6	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant T95A	Arabidopsis thaliana
72.8	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant N174A	Arabidopsis thaliana
480	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant S121A	Arabidopsis thaliana
779	-	NADPH	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant T95A	Arabidopsis thaliana
2870	-	glyoxylate	pH 7.8, temperature not specified in the publication, recombinant wild-type enzyme, value determined with the use of a double beam spectrophotometer	Arabidopsis thaliana
3407	-	glyoxylate	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant wild-type enzyme	Arabidopsis thaliana
4340	-	NADPH	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant F231A	Arabidopsis thaliana
10400	-	NADPH	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant D239A	Arabidopsis thaliana
10900	-	NADPH	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant N174A	Arabidopsis thaliana
24450	-	NADPH	pH 7.8, temperature not specified in the publication, recombinant wild-type enzyme, value determined with the use of a microplate reader	Arabidopsis thaliana
51700	-	NADPH	pH 7.8, temperature not specified in the publication, value determined with the use of a microplate reader, recombinant mutant S121A	Arabidopsis thaliana