Literature summary for 1.4.99.B4 extracted from

Watanabe, S.; Hiraoka, Y.; Endo, S.; Tanimoto, Y.; Tozawa, Y.; Watanabe, Y.
An enzymatic method to estimate the content of L-hydroxyproline (2015), J. Biotechnol., 199, 9-16 .

Search for more literature for 1.4.99.B4

Application

Application	Comment	Organism
analysis	estimation of the content of L-hydroxyprolines using coupling systems with metabolic enzymes of the trans-4-hydroxy-L-proline pathway (hydroxyproline 2-epimerase (HypE) and cis-4-hydroxy-D-proline dehydrogenase (HypDH)) and the trans-3-hydroxy-L-proline pathway (trans-3-hydroxy-L-proline dehydratase (T3LHypD) and DELTA1-pyrroline-2-carboxylate reductase (Pyr2CR)) from microorganisms. A functional expression system of recombinant HypDH with a heterooligomeric structure is constructed in Escherichia coli cells. Enzymological characterization reveals that the beta-subunit acts as a catalytic subunit, and also that assembly with other subunit(s) improves the kinetics for cis-4-hydroxy-D-proline and thermostability. By using a spectrophotometric assay with different wavelengths, the contents of trans-4-hydroxy-L-proline and trans-3-hydroxy-L-proline are successfully estimated within the ranges of 0.004-1 mM and 0.05-1 mM, respectively, and are consistent with the contents determined by HPLC. This enzymatic method is used to measure the content of trans-4-hydroxy-L-proline in the acid-hydrolysate of collagen, and blood plasma	Azospirillum brasilense

Cloned(Commentary)

Cloned (Comment)	Organism
cloning of the AbLhpB, AbLhpE and AbLhpF genes into different plasmid vectors, construction of a functional expression system of recombinant HypDH with a heterooligomeric structure in Escherichia coli cells	Azospirillum brasilense

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.00814	-	trans-4-hydroxy-D-proline	pH 8.0, 30°C, AbLhpBEF protein	Azospirillum brasilense
0.0224	-	D-proline	pH 8.0, 30°C, AbLhpBEF protein	Azospirillum brasilense
0.0628	-	cis-4-hydroxy-D-proline	pH 8.0, 30°C, AbLhpBEF protein	Azospirillum brasilense

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
cis-4-hydroxy-D-proline + acceptor	Azospirillum brasilense	the enzyme is involved in the metabolic pathway of trans-4-hydroxy-L-proline	(4R)-DELTA1-pyrroline-4-hydroxy-2-carboxylate + reduced acceptor	-	?

Organism

Organism	UniProt	Comment	Textmining
Azospirillum brasilense	S6C8W7 AND S6CFJ1 AND S6CLS6	S6C8W7: beta-subunit (catalytic subunit, AbLhpB), S6CFJ1: alpha-subunit (AbLhpE), S6CLS6: gamma-subunit (AbLhpF)	-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
1.02	-	substrate: D-proline, pH 8.0, 30°C, AbLhpBEF protein	Azospirillum brasilense
2.98	-	substrate: cis-4-hydroxy-D-proline, pH 8.0, 30°C, AbLhpBEF protein	Azospirillum brasilense

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
cis-4-hydroxy-D-proline + acceptor	the enzyme is involved in the metabolic pathway of trans-4-hydroxy-L-proline	Azospirillum brasilense	(4R)-DELTA1-pyrroline-4-hydroxy-2-carboxylate + reduced acceptor	-	?
cis-4-hydroxy-D-proline + acceptor	the Vmax/Km value of the enzyme for cis-4-hydroxy-D-proline is about 124fold and about 736fold higher than the Vmax/Km values for trans-4-hydroxy-D-proline and D-proline, respectively. Both AbLhpBEF (heteromer of alpha-, beta- and gamma-subunit) and AbLhpB (catalytic beta-subunit) are strictly specific to cis-4-hydroxy-D-proline	Azospirillum brasilense	(4R)-DELTA1-pyrroline-4-hydroxy-2-carboxylate + reduced acceptor	-	?
D-proline + acceptor	the Vmax/Km value of the enzyme for cis-4-hydroxy-D-proline is about 124fold and about 736fold higher than the Vmax/Km values for trans-4-hydroxy-D-proline and D-proline, respectively. Both AbLhpBEF (heteromer of alpha-, beta- and gamma-subunit) and AbLhpB (catalytic beta-subunit) are strictly specific to cis-4-hydroxy-D-proline	Azospirillum brasilense	?	-	?
trans-4-hydroxy-D-proline + acceptor	the Vmax/Km value of the enzyme for cis-4-hydroxy-D-proline is about 124fold and about 736fold higher than the Vmax/Km values for trans-4-hydroxy-D-proline and D-proline, respectively. Both AbLhpBEF (heteromer of alpha-, beta- and gamma-subunit) and AbLhpB (catalytic beta-subunit) are strictly specific to cis-4-hydroxy-D-proline	Azospirillum brasilense	?	-	?

Subunits

Subunits	Comment	Organism
dodecamer	alpha4beta4gamma4, the gamma-subunit significantly contributes to stabilizing the alpha4beta4gamma4 structure (and the alpha-subunit), but not to enzyme catalysis	Azospirillum brasilense

Synonyms

Synonyms	Comment	Organism
AbLhpBEF	-	Azospirillum brasilense
C4DHyp dehydrogenase	-	Azospirillum brasilense
HYPDH	-	Azospirillum brasilense
LhpBEF	-	Azospirillum brasilense

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Azospirillum brasilense

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
40	-	10 min, 70% loss of activity of the AbLhpB protein (catalytic subunit)	Azospirillum brasilense
50	-	10 min, the AbLhpBEF protein is stable, the AbLhpB protein (catalytic subunit) completely loses activity	Azospirillum brasilense

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
9	-	assay at	Azospirillum brasilense

Cofactor

Cofactor	Comment	Organism	Structure
FAD	-	Azospirillum brasilense

General Information

General Information	Comment	Organism
metabolism	the enzyme is involved in the metabolic pathway of trans-4-hydroxy-L-proline	Azospirillum brasilense

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Release 2023.1 (January 2023)