Literature summary for 2.6.1.44 extracted from

Dindo, M.; Grottelli, S.; Annunziato, G.; Giardina, G.; Pieroni, M.; Pampalone, G.; Faccini, A.; Cutruzzola, F.; Laurino, P.; Costantino, G.; Cellini, B.
Cycloserine enantiomers are reversible inhibitors of human alanine glyoxylate aminotransferase implications for primary hyperoxaluria type 1 (2019), Biochem. J., 476, 3751-3768 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene AGT, recombinant expression of wild-type and mutant G41R enzymes in Escherichia coli strain BL21, recombinant expression of wild-type AGT and the G41R variant in CHO-GO cells, that overexpress glycolate oxidase	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant wild-type enzyme in complex with inhibitors L- and D-cycloserine (LCS and DCS), hanging drop vapor diffusion technique, mixing of 0.001 ml of protein/ligand solution containing 0.215 mM enzyme, 2 mM DCS, and 50 mM potassium phosphate, pH 7.4, with an equal volume of reservoir solution containing 10-12% PEG 6000, 5% 2-methyl-2,4-pentanediol (MPD), and 0.1 M MES, pH 6.5, single crystals are cryoprotected by fast soaking into a reservoir solution containing 2 mM DCS and 25% MPD, for the AGT-LCS complex, AGT native crystals grown in the described conditions, with the exception of DCS, are soaked into a solution containing the reservoir and 20 mM LCS and 20% MPD, X-ray diffraction structure determination and analysis at 2.7-3.0 A resolution, modelling	Homo sapiens

Crystallization (Comment)

Organism

purified recombinant wild-type enzyme in complex with inhibitors L- and D-cycloserine (LCS and DCS), hanging drop vapor diffusion technique, mixing of 0.001 ml of protein/ligand solution containing 0.215 mM enzyme, 2 mM DCS, and 50 mM potassium phosphate, pH 7.4, with an equal volume of reservoir solution containing 10-12% PEG 6000, 5% 2-methyl-2,4-pentanediol (MPD), and 0.1 M MES, pH 6.5, single crystals are cryoprotected by fast soaking into a reservoir solution containing 2 mM DCS and 25% MPD, for the AGT-LCS complex, AGT native crystals grown in the described conditions, with the exception of DCS, are soaked into a solution containing the reservoir and 20 mM LCS and 20% MPD, X-ray diffraction structure determination and analysis at 2.7-3.0 A resolution, modelling

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
G41R	the naturally occuring missense mutation causes AGT misfolding, which induces aggregation and proteolytic degradation. Enzyme inhibitor D-cycloserin significantly improves the glyoxylate detoxification ability of CHO-GO cells expressing the enzyme mutant G41R variant, because it increases cell viability upon glycolate treatment. These data confirm that the treatment increases the amount of intraperoxisomal functional AGT able to metabolize glyoxylate	Homo sapiens

Inhibitors

Inhibitors	Comment	Organism
D-cycloserine	DCS, commercialized as Seromycin, a reversible inhibitor of AGT. DCS displays a time-dependent binding mainly generating an oxime intermediate, inhibition mechanism, overview	Homo sapiens
L-Cycloserine	LCS, a reversible inhibitor of AGT. LCS undergoes half-transamination generating a ketimine intermediate and behaves as a classical competitive inhibitor, inhibition mechanism, overview	Homo sapiens
additional information	the cycloserine enantiomers are reversible inhibitors of human alanine:glyoxylate aminotransferase. DCS, but not LCS, is able to promote the correct folding of the G41R variant, as revealed by its increased specific activity and expression as a soluble protein. This effect also translates into an increased glyoxylate detoxification ability of cells expressing the variant upon treatment with DCS. DCS might play a role as pharmacological chaperone. Inhibitor docking study and mass spectrometric analysis, identification of the wild-type and mutant AGT-cycloserine reaction products. Inhibitor effects on recombinant wild-type AGT and the G41R variant in CHO-GO cells, that overexpress glycolate oxidase, overview. Cell survival of CHO-GO-AGT-wild-type cells is much higher than that of CHO-GO-AGT-G41R mutant cells	Homo sapiens

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	steady-state Michaelis-Menten kinetics	Homo sapiens

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
peroxisome	-	Homo sapiens	5777	-

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
L-alanine + glyoxylate	Homo sapiens	-	pyruvate + glycine	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	P21549	-	-

Source Tissue

Source Tissue	Comment	Organism	Textmining
liver	-	Homo sapiens	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
L-alanine + glyoxylate	-	Homo sapiens	pyruvate + glycine	-	?

Subunits

Subunits	Comment	Organism
homodimer	-	Homo sapiens

Synonyms

Synonyms	Comment	Organism
AGT	-	Homo sapiens
alanine:glyoxylate aminotransferase	-	Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Homo sapiens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.4	-	assay at	Homo sapiens

Cofactor

Cofactor	Comment	Organism	Structure
pyridoxal 5'-phosphate	PLP	Homo sapiens

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism	Structure
additional information	-	additional information	inhibition kinetics	Homo sapiens

General Information

General Information	Comment	Organism
evolution	AGT is homodimeric and belongs to the fold type I family of PLP-dependent enzymes	Homo sapiens
malfunction	deficit of AGT leads to primary hyperoxaluria type I (PH1), a rare disease characterized by calcium oxalate stones deposition in the urinary tract as a consequence of glyoxylate accumulation. Most missense mutations cause AGT misfolding, as in the case of the G41R, which induces aggregation and proteolytic degradation	Homo sapiens
additional information	the AGT catalytic mechanism is typical of PLP-dependent aminotransferases and comprises two half-reactions. In the first one, the alpha-amino group of the substrate L-alanine displaces the epsilon-amino group of Lys209 producing the external aldimine. Then, Lys209 acts as a general base for the 1,3-prototropic shift generating a ketimine intermediate, which hydrolyzes to pyruvate and pyridoxamine 5'-phosphate (PMP). In the second half-reaction, glyoxylate binds to AGT-PMP and, through the same steps of the first reaction but in a reverse order, is converted to glycine regenerating AGT-PLP	Homo sapiens
physiological function	alanine:glyoxylate aminotransferase (AGT) catalyzes the conversion of L-alanine and glyoxylate into pyruvate and glycine in liver peroxisomes, using pyridoxal 5'-phosphate (PLP) as coenzyme	Homo sapiens