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 (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 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 | 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 | 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 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 | 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 | 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 | Comment | Organism |
---|---|---|
AbLhpBEF | - |
Azospirillum brasilense |
C4DHyp dehydrogenase | - |
Azospirillum brasilense |
HYPDH | - |
Azospirillum brasilense |
LhpBEF | - |
Azospirillum brasilense |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Azospirillum brasilense |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
9 | - |
assay at | Azospirillum brasilense |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | - |
Azospirillum brasilense |
General Information | Comment | Organism |
---|---|---|
metabolism | the enzyme is involved in the metabolic pathway of trans-4-hydroxy-L-proline | Azospirillum brasilense |