Literature summary for 2.6.1.52 extracted from

Sekula, B.; Ruszkowski, M.; Dauter, Z.
Structural analysis of phosphoserine aminotransferase (isoform 1) from Arabidopsis thaliana - the enzyme involved in the phosphorylated pathway of serine biosynthesis (2018), Front. Plant Sci., 9, 876 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene PSAT1, recombinant expression of His6-tagged in Escherichia coli strain BL21 Gold	Arabidopsis thaliana

Crystallization (Commentary)

Crystallization (Comment)	Organism
three crystal structures of isozyme AtPSAT1 are captured at different stages of the reaction: (i) internal aldimine state with PLP covalently bound to the catalytic K265, (ii) holoenzyme in complex with pyridoxamine-50-phosphate (PMP) after transfer of the amino group from glutamate and (iii) the geminal diamine intermediate state wherein the cofactor is covalently bound to both, K265 and PSer. These snapshots over the course of the reaction present the detailed architecture of AtPSAT1. Purified detagged recombinant enzyme in complex with cofactor PLP or pyridoxamine or product phosphoserine, sitting drop method, mixing of 15 mg/ml protein and 1 mM ligand (PLP, PMP) or 50 mM ligand (PSer) in 25 mM HEPES, pH 7.4, 100 mM KCl, 50 mM NaCl, and 1 mM TCEP with crystallization solution containing 0.2 M lithium sulfate, 17% PEG 3350, and 0.1 M Tris at pH 8.5 for ATPSAT1-PLP, with 0.2 M lithium sulfate, 17% PEG 3350, and 0.1 M Tris at pH 8.5, and 10 mM glutamate for AtPSAT1-PMP, and with 19% PEG 3350 and 0.1 M Tris at pH 8.5 for AtPSAT1-PSer, xadX-ray diffraction structure determination and analysis at 1.57-1.70 A resolution	Arabidopsis thaliana

Crystallization (Comment)

Organism

three crystal structures of isozyme AtPSAT1 are captured at different stages of the reaction: (i) internal aldimine state with PLP covalently bound to the catalytic K265, (ii) holoenzyme in complex with pyridoxamine-50-phosphate (PMP) after transfer of the amino group from glutamate and (iii) the geminal diamine intermediate state wherein the cofactor is covalently bound to both, K265 and PSer. These snapshots over the course of the reaction present the detailed architecture of AtPSAT1. Purified detagged recombinant enzyme in complex with cofactor PLP or pyridoxamine or product phosphoserine, sitting drop method, mixing of 15 mg/ml protein and 1 mM ligand (PLP, PMP) or 50 mM ligand (PSer) in 25 mM HEPES, pH 7.4, 100 mM KCl, 50 mM NaCl, and 1 mM TCEP with crystallization solution containing 0.2 M lithium sulfate, 17% PEG 3350, and 0.1 M Tris at pH 8.5 for ATPSAT1-PLP, with 0.2 M lithium sulfate, 17% PEG 3350, and 0.1 M Tris at pH 8.5, and 10 mM glutamate for AtPSAT1-PMP, and with 19% PEG 3350 and 0.1 M Tris at pH 8.5 for AtPSAT1-PSer, xadX-ray diffraction structure determination and analysis at 1.57-1.70 A resolution

Arabidopsis thaliana

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
chloroplast	isozyme PSAT1 has a truncated 71-residue-long chloroplast targeting signal peptide	Arabidopsis thaliana	9507	-

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
80000	-	about, detagged recombinant enzyme, gel filtration	Arabidopsis thaliana

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
O-phospho-L-serine + 2-oxoglutarate	Arabidopsis thaliana	-	3-phosphooxypyruvate + L-glutamate	-	r

Organism

Organism	UniProt	Comment	Textmining
Arabidopsis thaliana	Q96255	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-tagged from Escherichia coli strain BL21 Gold by nickel affinity chromatography, tag cleavage through TEV protease, dialysis, gel filtration and ultrafiltration	Arabidopsis thaliana

Reaction

Reaction	Comment	Organism	Reaction ID
O-phospho-L-serine + 2-oxoglutarate = 3-phosphooxypyruvate + L-glutamate	bimolecular ping-pong mechanism, the transamination reaction catalyzed by PSAT consists of two reversible half-reactions, detailed overview	Arabidopsis thaliana	a

Source Tissue

Source Tissue	Comment	Organism	Textmining
leaf	-	Arabidopsis thaliana	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	the transamination reaction catalyzed by PSAT consists of two reversible half-reactions, overview. Substrate binding requires a conformational change of AtPSAT1	Arabidopsis thaliana	?	-	-
O-phospho-L-serine + 2-oxoglutarate	-	Arabidopsis thaliana	3-phosphooxypyruvate + L-glutamate	-	r

Subunits

Subunits	Comment	Organism
homodimer	PSAT isoform 1 is a dimeric S-shaped protein	Arabidopsis thaliana
More	the crystal asymmetric unit with four subunits represents two functional AtPSAT1 dimers. The subunit of AtPSAT1 consists of two domains with mixed alpha/beta topology. This is a typical fold of class IV of the alpha-type aminotransferases with a much larger N-terminal catalytic domain and a smaller C-terminal domain. Hydrophobic core of the N-terminal domain is constituted by a seven-stranded beta-sheet with one anti-parallel strand. Subunit structure model, overview	Arabidopsis thaliana

Synonyms

Synonyms	Comment	Organism
AtPSAT1	-	Arabidopsis thaliana
phosphoserine aminotransferase	-	Arabidopsis thaliana
phosphoserine aminotransferase 1	-	Arabidopsis thaliana
PSAT	-	Arabidopsis thaliana
PSAT1	-	Arabidopsis thaliana

Cofactor

Cofactor	Comment	Organism	Structure
pyridoxal 5'-phosphate	PLP, dependent on, the PLP prosthetic group creates a Schiff base (internal aldimine) between C4' and Nzeta of K265, PLP bound in the active site forms an internal aldimine with residue K265, the residue placed in the coil between beta9 and beta10	Arabidopsis thaliana

General Information

General Information	Comment	Organism
evolution	phosphoserine aminotransferases belong to the class IV of aminotransferases with the alpha-type fold. In general, this class of enzymes is characterized by the presence of two domains with mixed alpha/beta fold	Arabidopsis thaliana
metabolism	phosphoserine aminotransferase (PSAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the conversion of 3-phosphohydroxypyruvate (3-PHP) to 3-phosphoserine (PSer) in an L-glutamate (Glu)-linked reversible transamination reaction. This process in plants takes place in plastids. It is a part of the phosphorylated pathway of serine biosynthesis, one of three routes recognized in plant organisms that yield serine. In this three-step biotransformation, 3-phosphoglycerate (3-PGA) delivered from plastidial glycolysis and Calvin cycle is oxidized by 3-PGA dehydrogenase. Then, 3-PHP is subjected to transamination with Glu to yield PSer and 2-oxoglutarate (AKG). In the last step of the pathway, serine is produced by the action of phosphoserine phosphatase	Arabidopsis thaliana
additional information	conformational changes of the protein during the catalytic event concern (i) the neighborhood of K265 when the amino group is transferred to the cofactor to form PMP and (ii) movement of the gate-keeping loop (residues 391-401) upon binding of of 3-phosphohydroxypyruvate to 3-phosphoserine. The latter conformational change of the loop may likely be one of key elements that regulate catalytic activity of PSATs. The conserved catalytic lysine, which directly follows a hydrophobic beta-strand, is localized closer to the C-terminus than the Gly-rich region. PSATs have an aspartate residue which hydrogen bonds the pyridoxal ring N1 atom and precedes the Schiff base lysine by 20-50 amino acids. The complex structure with pyridoxamine shows the enzyme primes for a covalent binding of 3-phosphohydroxypyruvate. The complex structure with phopshoserine reveals the geminal diamine intermediate state	Arabidopsis thaliana