Literature summary for 5.1.1.10 extracted from

Kuan, Y.; Thiruvengadam, V.; Lin, J.; Liu, J.; Chen, T.; Wu, H.; Wang, W.; Chen, L.
Broad-specificity amino acid racemase, a novel non-antibiotic selectable marker for transgenic plants (2018), Plant Biotechnol. Rep., 12, 27-38 .

No PubMed abstract available

Application

Application	Comment	Organism
diagnostics	broad-specificity amino acid racemase is a non-antibiotic selectable marker for transgenic plants and can be used in the development of genetically modified plants without using antibiotic resistance marker genes. Selection with different amino acid enantiomers using Bsar, Bsar-R174A and Bsar-R174K gene as selectable markers, overview	Pseudomonas putida

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant overexpression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 (DE3) and complementation of lysine or asparagine auxotroph strains (Escherichia coli lysine auxotrophic strain BCRC 51734 and asparagine auxotrophic strain CGSC 4813) recombinant expression of wild-type and mutant enzymes in Arabidopsis thaliana ecotype Columbia and Oryza sativa cv. Tainung 67 plants using the Agrobacterium tumefaciens strain EHA105 transformation system. The Bsar variant, Bsar-R174K, is useful as a selectable marker gene in Arabidopsis and rice that are susceptible to L-lysine and D-alanine. The transformation of Arabidopsis thalinana with Bsar or Bsar variants based on D-alanine selection reveals that Bsar-R174K has the greatest efficiency (2.40%), superior to kanamycin selection-based transformation (1.10%). L-lysine-based selection exhibits lower efficiency for Bsar-R174K (0.17%). The progenies of selected Bsar-R174K transgenic Arabidopsis thaliana plants reveals normal growth properties. Bsar-R174K mutant transgenic Oryza sativa is obtained on L-lysine medium with an efficiency of 0.9%, and the progenies of the transgenic rice show morphologically normal phenotypes comparable with their wild-type counterparts. RT-PCR expression analysis	Pseudomonas putida

Cloned (Comment)

Organism

recombinant overexpression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 (DE3) and complementation of lysine or asparagine auxotroph strains (Escherichia coli lysine auxotrophic strain BCRC 51734 and asparagine auxotrophic strain CGSC 4813) recombinant expression of wild-type and mutant enzymes in Arabidopsis thaliana ecotype Columbia and Oryza sativa cv. Tainung 67 plants using the Agrobacterium tumefaciens strain EHA105 transformation system. The Bsar variant, Bsar-R174K, is useful as a selectable marker gene in Arabidopsis and rice that are susceptible to L-lysine and D-alanine. The transformation of Arabidopsis thalinana with Bsar or Bsar variants based on D-alanine selection reveals that Bsar-R174K has the greatest efficiency (2.40%), superior to kanamycin selection-based transformation (1.10%). L-lysine-based selection exhibits lower efficiency for Bsar-R174K (0.17%). The progenies of selected Bsar-R174K transgenic Arabidopsis thaliana plants reveals normal growth properties. Bsar-R174K mutant transgenic Oryza sativa is obtained on L-lysine medium with an efficiency of 0.9%, and the progenies of the transgenic rice show morphologically normal phenotypes comparable with their wild-type counterparts. RT-PCR expression analysis

Pseudomonas putida

Protein Variants

Protein Variants	Comment	Organism
R174A	site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme	Pseudomonas putida
R174K	site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme. Mutant Bsar-R174K shows no deleterious effects on the transgenic plants and broad range amino acid racemase activity, might serve as a good selection marker	Pseudomonas putida

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
L-alanine	Pseudomonas putida	-	D-alanine	-	r
L-alanine	Pseudomonas putida DSM 84	-	D-alanine	-	r
L-arginine	Pseudomonas putida	-	D-arginine	-	r
L-arginine	Pseudomonas putida DSM 84	-	D-arginine	-	r
L-asparagine	Pseudomonas putida	-	D-asparagine	-	r
L-lysine	Pseudomonas putida	-	D-lysine	-	r
L-lysine	Pseudomonas putida DSM 84	-	D-lysine	-	r
L-Methionine	Pseudomonas putida	-	D-Methionine	-	r
L-Methionine	Pseudomonas putida DSM 84	-	D-Methionine	-	r
L-serine	Pseudomonas putida	-	D-serine	-	r
L-serine	Pseudomonas putida DSM 84	-	D-serine	-	r

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas putida	-	-	-
Pseudomonas putida DSM 84	-	-	-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
0.1	-	purified recombinant mutant R174A, pH 7.0, 37°C, substrate L-serine	Pseudomonas putida
0.12	-	purified recombinant mutant R174K, pH 7.0, 37°C, substrate L-alanine	Pseudomonas putida
0.12	-	purified recombinant mutant R174K, pH 7.0, 37°C, substrate L-methionine	Pseudomonas putida
0.15	-	purified recombinant mutant R174A, pH 7.0, 37°C, substrate L-asparagine	Pseudomonas putida
0.3	-	purified recombinant mutant R174K, pH 7.0, 37°C, substrate L-serine	Pseudomonas putida
0.36	-	purified recombinant mutant R174A, pH 7.0, 37°C, substrate L-methionine	Pseudomonas putida
0.62	-	purified recombinant mutant R174A, pH 7.0, 37°C, substrate L-arginine	Pseudomonas putida
0.64	-	purified recombinant mutant R174A, pH 7.0, 37°C, substrate L-lysine	Pseudomonas putida
0.82	-	purified recombinant mutant R174K, pH 7.0, 37°C, substrate L-asparagine	Pseudomonas putida
2.46	-	purified recombinant mutant R174K, pH 7.0, 37°C, substrate L-lysine	Pseudomonas putida
3.22	-	purified recombinant mutant R174K, pH 7.0, 37°C, substrate L-arginine	Pseudomonas putida
7.5	-	purified recombinant wild-type enzyme, pH 7.0, 37°C, substrate L-asparagine	Pseudomonas putida
56	-	purified recombinant wild-type enzyme, pH 7.0, 37°C, substrate L-alanine	Pseudomonas putida
209	-	purified recombinant wild-type enzyme, pH 7.0, 37°C, substrate L-serine	Pseudomonas putida
256	-	purified recombinant wild-type enzyme, pH 7.0, 37°C, substrate L-methionine	Pseudomonas putida
1224	-	purified recombinant wild-type enzyme, pH 7.0, 37°C, substrate L-arginine	Pseudomonas putida
1423	-	purified recombinant wild-type enzyme, pH 7.0, 37°C, substrate L-lysine	Pseudomonas putida

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
L-alanine	-	Pseudomonas putida	D-alanine	-	r
L-alanine	-	Pseudomonas putida DSM 84	D-alanine	-	r
L-arginine	-	Pseudomonas putida	D-arginine	-	r
L-arginine	-	Pseudomonas putida DSM 84	D-arginine	-	r
L-asparagine	-	Pseudomonas putida	D-asparagine	-	r
L-lysine	-	Pseudomonas putida	D-lysine	-	r
L-lysine	-	Pseudomonas putida DSM 84	D-lysine	-	r
L-Methionine	-	Pseudomonas putida	D-Methionine	-	r
L-Methionine	-	Pseudomonas putida DSM 84	D-Methionine	-	r
L-serine	-	Pseudomonas putida	D-serine	-	r
L-serine	-	Pseudomonas putida DSM 84	D-serine	-	r

Synonyms

Synonyms	Comment	Organism
broad-specificity amino acid racemase	-	Pseudomonas putida
Bsar	-	Pseudomonas putida

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Pseudomonas putida

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	assay at	Pseudomonas putida

Cofactor

Cofactor	Comment	Organism	Structure
pyridoxal 5'-phosphate	-	Pseudomonas putida