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Literature summary for 1.5.99.B2 extracted from

  • Serrano, H.; Blanchard, J.S.
    Kinetic and isotopic characterization of L-proline dehydrogenase from Mycobacterium tuberculosis (2013), Biochemistry, 52, 5009-5015 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
recombinant overexpression of C-terminally His6-tagged wild-type and mutant enzymes in Mycobacterium smegmatis mc24517 cells using vector pYUB1062 Mycobacterium tuberculosis

Protein Variants

Protein Variants Comment Organism
K110A site-directed mutagenesis, inactive mutant Mycobacterium tuberculosis
Y203F site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Mycobacterium tuberculosis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten kinetics, steady-state kinetics and pre-steady-state kinetics of wild-type and mutant enzymes, stopped-flow kinetic isotope effects, bisubstrate kinetic analysis, overview Mycobacterium tuberculosis
3.4
-
2,6-dichlorophenolindophenol pH 7.1, 25°C, recombinant wild-type enzyme Mycobacterium tuberculosis
5.7
-
L-proline pH 7.1, 25°C, recombinant wild-type enzyme Mycobacterium tuberculosis
860
-
L-proline pH 7.1, 25°C, recombinant mutant Y203F Mycobacterium tuberculosis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-proline + acceptor Mycobacterium tuberculosis
-
(S)-1-pyrroline-5-carboxylate + reduced acceptor
-
?
L-proline + acceptor Mycobacterium tuberculosis H37Rv
-
(S)-1-pyrroline-5-carboxylate + reduced acceptor
-
?

Organism

Organism UniProt Comment Textmining
Mycobacterium tuberculosis O50444
-
-
Mycobacterium tuberculosis H37Rv O50444
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant C-terminally His6-tagged wild-type and mutant enzymes from Mycobacterium smegmatis mc24517 cells by nickel affinity chromatography Mycobacterium tuberculosis

Reaction

Reaction Comment Organism Reaction ID
L-proline + acceptor = (S)-1-pyrroline-5-carboxylate + reduced acceptor ping-pong mechanism, the transfer of hydride from L-proline to FAD is rate-limiting for the reductive half-reaction, but the overall reaction is limited by reduced flavin reoxidation in the second half-reaction. Solvent and multiple kinetic isotope effects suggest that L-proline oxidation occurs in a stepwise rather than concerted mechanism Mycobacterium tuberculosis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-proline + 2,6-dichlorophenolindophenol artificial electron acceptor Mycobacterium tuberculosis (S)-1-pyrroline-5-carboxylate + reduced 2,6-dichlorophenolindophenol
-
?
L-proline + 2,6-dichlorophenolindophenol artificial electron acceptor Mycobacterium tuberculosis H37Rv (S)-1-pyrroline-5-carboxylate + reduced 2,6-dichlorophenolindophenol
-
?
L-proline + acceptor
-
Mycobacterium tuberculosis (S)-1-pyrroline-5-carboxylate + reduced acceptor
-
?
L-proline + acceptor
-
Mycobacterium tuberculosis H37Rv (S)-1-pyrroline-5-carboxylate + reduced acceptor
-
?

Synonyms

Synonyms Comment Organism
PRODH
-
Mycobacterium tuberculosis
proline dehydrogenase
-
Mycobacterium tuberculosis
prub
-
Mycobacterium tuberculosis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Mycobacterium tuberculosis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
33.5
-
L-proline pH 7.1, 25°C, recombinant wild-type enzyme Mycobacterium tuberculosis
100.6
-
L-proline pH 7.1, 25°C, recombinant mutant Y203F Mycobacterium tuberculosis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.1 7.4 assay at Mycobacterium tuberculosis

Cofactor

Cofactor Comment Organism Structure
FAD
-
Mycobacterium tuberculosis

General Information

General Information Comment Organism
metabolism proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme involved in the biosynthesis of L-glutamate Mycobacterium tuberculosis
additional information residue Y203 serves to bind and orient L-proline in the active site, residue K110 is the active site base observed in the pH profile Mycobacterium tuberculosis
physiological function the monofunctional proline dehydrogenase (ProDH) performs the flavin-dependent oxidation of L-proline to DELTA1-pyrroline-5-carboxylate in the proline catabolic pathway Mycobacterium tuberculosis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.12
-
L-proline pH 7.1, 25°C, recombinant mutant Y203F Mycobacterium tuberculosis
5.88
-
L-proline pH 7.1, 25°C, recombinant wild-type enzyme Mycobacterium tuberculosis