Literature summary for 5.1.1.10 extracted from

Kawakami, R.; Sakuraba, H.; Ohmori, T.; Ohshima, T.
First characterization of an archaeal amino acid racemase with broad substrate specificity from the hyperthermophile Pyrococcus horikoshii OT-3 (2017), J. Biosci. Bioeng., 124, 23-27 .

Application

Application	Comment	Organism
synthesis	the high reactivity toward Trp and Tyr, as well as extremely high thermostability, is likely a major advantage of using BAR for biochemical conversion of these aromatic amino acids	Pyrococcus horikoshii

Cloned(Commentary)

Cloned (Comment)	Organism
enzyme is produced mainly as an inclusion body in Escherichia coli. In this study, expression of the recombinant protein into the soluble fraction is markedly improved by coexpression with chaperone molecules	Pyrococcus horikoshii
recombinant expression of enzyme BAR in inclusion bodies in Escherichia coli strain BL21(DE3), expression of the recombinant protein in the soluble fraction is markedly improved by co-expression with chaperone molecules, i.e. chaperone proteins DnaK-DnaJ-GrpE and GroEL-GroES, GroELGroES and Tig, and Tig, method optimization	Pyrococcus horikoshii

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
1.68	-	D-Trp	pH 7.0, 80°C	Pyrococcus horikoshii
1.68	-	D-tryptophan	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
2.23	-	L-Tyr	pH 7.0, 80°C	Pyrococcus horikoshii
2.23	-	L-tyrosine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
2.77	-	D-Tyr	pH 7.0, 80°C	Pyrococcus horikoshii
2.77	-	D-tyrosine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
2.78	-	D-Phe	pH 7.0, 80°C	Pyrococcus horikoshii
2.78	-	D-phenylalanine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
3.41	-	L-Val	pH 7.0, 80°C	Pyrococcus horikoshii
3.41	-	L-valine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
3.94	-	L-Ile	pH 7.0, 80°C	Pyrococcus horikoshii
3.94	-	L-isoleucine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
4.19	-	D-Leu	pH 7.0, 80°C	Pyrococcus horikoshii
4.19	-	D-leucine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
4.28	-	L-Met	pH 7.0, 80°C	Pyrococcus horikoshii
4.28	-	L-methionine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
4.95	-	L-Leu	pH 7.0, 80°C	Pyrococcus horikoshii
4.95	-	L-leucine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
5.84	-	L-Trp	pH 7.0, 80°C	Pyrococcus horikoshii
5.84	-	L-tryptophan	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
7.86	-	D-Met	pH 7.0, 80°C	Pyrococcus horikoshii
7.86	-	D-methionine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
8.23	-	L-Phe	pH 7.0, 80°C	Pyrococcus horikoshii
8.23	-	L-phenylalanine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
9.54	-	D-Ile	pH 7.0, 80°C	Pyrococcus horikoshii
9.54	-	D-isoleucine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
9.82	-	D-Val	pH 7.0, 80°C	Pyrococcus horikoshii
9.82	-	D-valine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
35.6	-	D-Ala	pH 7.0, 80°C	Pyrococcus horikoshii
35.6	-	D-alanine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
56	-	L-Ala	pH 7.0, 80°C	Pyrococcus horikoshii
56	-	L-alanine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
91000	-	gel filtration	Pyrococcus horikoshii
91000	-	recombinant enzyme, gel filtration	Pyrococcus horikoshii

Organism

Organism	UniProt	Comment	Textmining
Pyrococcus horikoshii	O57878	-	-
Pyrococcus horikoshii OT-3	O57878	-	-

Purification (Commentary)

Purification (Comment)	Organism
-	Pyrococcus horikoshii
recombinant soluble enzyme BAR from Escherichia coli strain BL21(DE3) by heat treatment at 90°C for 20 min, ammonium sulfate fractionation, and hydrophobic interaction chromatography, dialysis, anion echange chromatography, and again dialysis	Pyrococcus horikoshii

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
D-Ala	-	Pyrococcus horikoshii	L-Ala	-	r
D-alanine	-	Pyrococcus horikoshii	L-alanine	-	r
D-Ile	-	Pyrococcus horikoshii	L-Ile	-	r
D-isoleucine	-	Pyrococcus horikoshii	L-isoleucine	-	r
D-Leu	-	Pyrococcus horikoshii	L-Leu	-	r
D-leucine	-	Pyrococcus horikoshii	L-leucine	-	r
D-Met	-	Pyrococcus horikoshii	L-Met	-	r
D-methionine	-	Pyrococcus horikoshii	L-methionine	-	r
D-Phe	-	Pyrococcus horikoshii	L-Phe	-	r
D-phenylalanine	-	Pyrococcus horikoshii	L-phenylalanine	-	r
D-serine	-	Pyrococcus horikoshii	L-serine	-	r
D-threonine	-	Pyrococcus horikoshii	L-threonine	-	r
D-Trp	-	Pyrococcus horikoshii	L-Trp	-	r
D-tryptophan	-	Pyrococcus horikoshii	L-tryptophan	-	r
D-Tyr	-	Pyrococcus horikoshii	L-Tyr	-	r
D-tyrosine	-	Pyrococcus horikoshii	L-tyrosine	-	r
D-Val	-	Pyrococcus horikoshii	L-Val	-	r
D-valine	-	Pyrococcus horikoshii	L-valine	-	r
L-Ala	-	Pyrococcus horikoshii	D-Ala	-	r
L-Ala	-	Pyrococcus horikoshii OT-3	D-Ala	-	r
L-alanine	-	Pyrococcus horikoshii	D-alanine	-	r
L-Ile	-	Pyrococcus horikoshii	D-Ile	-	r
L-isoleucine	-	Pyrococcus horikoshii	D-isoleucine	-	r
L-Leu	-	Pyrococcus horikoshii	D-Leu	-	r
L-Leu	-	Pyrococcus horikoshii OT-3	D-Leu	-	r
L-leucine	-	Pyrococcus horikoshii	D-leucine	-	r
L-Met	-	Pyrococcus horikoshii	D-Met	-	r
L-Met	-	Pyrococcus horikoshii OT-3	D-Met	-	r
L-Methionine	-	Pyrococcus horikoshii	D-Methionine	-	r
L-Phe	-	Pyrococcus horikoshii	D-Phe	-	r
L-phenylalanine	-	Pyrococcus horikoshii	D-phenylalanine	-	r
L-serine	-	Pyrococcus horikoshii	D-serine	-	r
L-threonine	-	Pyrococcus horikoshii	D-threonine	-	r
L-Trp	-	Pyrococcus horikoshii	D-Trp	-	r
L-tryptophan	-	Pyrococcus horikoshii	D-tryptophan	-	r
L-Tyr	-	Pyrococcus horikoshii	D-Tyr	-	r
L-tyrosine	-	Pyrococcus horikoshii	D-tyrosine	-	r
L-Val	-	Pyrococcus horikoshii	D-Val	-	r
L-valine	-	Pyrococcus horikoshii	D-valine	-	r
additional information	the enzyme predominantly shows activity toward hydrophobic and aromatic amino acids. No activity toward Arg, His, Gln, and Asn	Pyrococcus horikoshii	?	-	?
additional information	the enzyme BAR has broad substrate specificity, it is active toward Met, Leu, Phe, Ile, Val, Ala, Trp, Tyr, Thr, and Ser, but no activity toward Pro, Asp, Glu, Arg, His, Gln, Cys, Lys, and Asn. In particular, the high reaction rate with Trp and Tyr, in addition to Leu, Met and Phe as substrates is a noteworthy feature of this enzyme. Histochemic analysis of D- and L-amino acid products. The Vmax values obtained with Ala, Val, Ile, Leu, and Met in D-amino acid forming reactions are lower than in L-amino acid forming reactions. By contrast, the Vmax values for Phe, Tyr, and Trp in D-amino acid forming reactions are higher than in L-amino acid forming reaction. The D-forms of Arg, His, Asn, and Gln are not quantitatively detected	Pyrococcus horikoshii	?	-	?
additional information	the enzyme predominantly shows activity toward hydrophobic and aromatic amino acids. No activity toward Arg, His, Gln, and Asn	Pyrococcus horikoshii OT-3	?	-	?
additional information	the enzyme BAR has broad substrate specificity, it is active toward Met, Leu, Phe, Ile, Val, Ala, Trp, Tyr, Thr, and Ser, but no activity toward Pro, Asp, Glu, Arg, His, Gln, Cys, Lys, and Asn. In particular, the high reaction rate with Trp and Tyr, in addition to Leu, Met and Phe as substrates is a noteworthy feature of this enzyme. Histochemic analysis of D- and L-amino acid products. The Vmax values obtained with Ala, Val, Ile, Leu, and Met in D-amino acid forming reactions are lower than in L-amino acid forming reactions. By contrast, the Vmax values for Phe, Tyr, and Trp in D-amino acid forming reactions are higher than in L-amino acid forming reaction. The D-forms of Arg, His, Asn, and Gln are not quantitatively detected	Pyrococcus horikoshii OT-3	?	-	?

Subunits

Subunits	Comment	Organism
dimer	2 * 52100, SDS-PAGE	Pyrococcus horikoshii
homodimer	2 * 55800, recombinant enzyme, SDS-PAGE, 2 * 52100, about, sequence calculation	Pyrococcus horikoshii

Synonyms

Synonyms	Comment	Organism
AAR	-	Pyrococcus horikoshii
amino acid racemase	-	Pyrococcus horikoshii
BAR	-	Pyrococcus horikoshii
broad substrate specificity amino acid racemase	-	Pyrococcus horikoshii
PH0138	-	Pyrococcus horikoshii

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
80	-	assay at	Pyrococcus horikoshii
95	-	-	Pyrococcus horikoshii
95	-	or above	Pyrococcus horikoshii

Temperature Range [°C]

Temperature Minimum [°C]	Temperature Maximum [°C]	Comment	Organism
50	80	the relative activities at 50°C and 80°C are 12% and 70%, respectively, as compared with that at 95°C	Pyrococcus horikoshii
50	95	activity range, the relative activities at 50°C and 80°C are 12% and 70%, respectively, as compared with that at 95°C	Pyrococcus horikoshii

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
80	-	enzyme retains its full activity after incubation at 80°C for at least 2 h in buffer pH 7-10	Pyrococcus horikoshii
80	-	purified enzyme protein at 80°C for 2 h under various pH conditions, BAR shows no loss of activity at pHs ranging from pH 7.0 to pH 10.0. At pH 7.0, the enzyme retains its full activity, even after incubation at 80°C for 24 h	Pyrococcus horikoshii

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6.5	7.5	at a temperature of 60°C, the highest activity is detected at around pH 6.5-7.5	Pyrococcus horikoshii
6.5	7.5	at 60°C, the highest activity is detected at around pH 6.5-7.5	Pyrococcus horikoshii
7	-	assay at	Pyrococcus horikoshii

pH Range

pH Minimum	pH Maximum	Comment	Organism
6	10	over 80% of maximal activity within this range, about 40% at pH 4.0, and about 50% at pH 11.0	Pyrococcus horikoshii

Cofactor

Cofactor	Comment	Organism	Structure
pyridoxal 5'-phosphate	dependent on	Pyrococcus horikoshii

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.159	-	L-Ala	pH 7.0, 80°C	Pyrococcus horikoshii
0.159	-	L-alanine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
0.75	-	D-alanine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
0.95	-	D-Ala	pH 7.0, 80°C	Pyrococcus horikoshii
1.31	-	D-Trp	pH 7.0, 80°C	Pyrococcus horikoshii
1.31	-	D-tryptophan	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
1.97	-	L-Val	pH 7.0, 80°C	Pyrococcus horikoshii
1.97	-	L-valine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
2.39	-	L-Trp	pH 7.0, 80°C	Pyrococcus horikoshii
2.39	-	L-tryptophan	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
2.67	-	D-Val	pH 7.0, 80°C	Pyrococcus horikoshii
2.67	-	D-valine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
4.12	-	D-Ile	pH 7.0, 80°C	Pyrococcus horikoshii
4.12	-	D-isoleucine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
5.5	-	L-Ile	pH 7.0, 80°C	Pyrococcus horikoshii
5.5	-	L-isoleucine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
5.69	-	D-Tyr	pH 7.0, 80°C	Pyrococcus horikoshii
5.69	-	D-tyrosine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
8.26	-	L-Met	pH 7.0, 80°C	Pyrococcus horikoshii
8.26	-	L-methionine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
11	-	L-Phe	pH 7.0, 80°C	Pyrococcus horikoshii
11	-	L-phenylalanine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
11.4	-	L-Tyr	pH 7.0, 80°C	Pyrococcus horikoshii
11.4	-	L-tyrosine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
11.9	-	L-Leu	pH 7.0, 80°C	Pyrococcus horikoshii
11.9	-	L-leucine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
12.7	-	D-Met	pH 7.0, 80°C	Pyrococcus horikoshii
12.7	-	D-methionine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
18.4	-	D-Leu	pH 7.0, 80°C	Pyrococcus horikoshii
18.4	-	D-leucine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii
19.9	-	D-Phe	pH 7.0, 80°C	Pyrococcus horikoshii
19.9	-	D-phenylalanine	pH 7.0, 80°C, recombinant enzyme	Pyrococcus horikoshii