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Literature summary for 1.14.13.81 extracted from

  • Chen, G.E.; Adams, N.B.P.; Jackson, P.J.; Dickman, M.J.; Hunter, C.N.
    How the O2-dependent Mg-protoporphyrin monomethyl ester cyclase forms the fifth ring of chlorophylls (2021), Nat. plants, 7, 365-375 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
gene acsF, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain C43(DE3) Rubrivivax gelatinosus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information steady-state kinetics. The dependence of the initial rate on MgPME followed Michaelis-Menten kinetics, a sigmoidal relationship is found between the initial rate and the NADPH concentration so the Hill equation is used to fit the kinetic data Rubrivivax gelatinosus
0.0073
-
magnesium-protoporphyrin IX 13-monomethyl ester Michaelis-Menten kinetics, recombinant enzyme, pH 7.7, 30┬░C Rubrivivax gelatinosus
0.16
-
magnesium-protoporphyrin IX 13-monomethyl ester sigmoidal kinetics, recombinant enzyme, pH 7.7, 30┬░C Rubrivivax gelatinosus

Metals/Ions

Metals/Ions Comment Organism Structure
Fe2+ required, presence of a diiron cluster in AcsF Rubrivivax gelatinosus

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
210000
-
detergent solubilized, purified recombinant His6-tagged enzyme, gel filtration Rubrivivax gelatinosus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
magnesium-protoporphyrin IX 13-monomethyl ester + 3 NADPH + 3 H+ + 3 O2 Rubrivivax gelatinosus overall reaction 3,8-divinyl protochlorophyllide a + 3 NADP+ + 5 H2O
-
?

Organism

Organism UniProt Comment Textmining
Rubrivivax gelatinosus P0DJN9
-
-

Purification (Commentary)

Purification (Comment) Organism
single-subunit O2-dependent cyclase AcsF, recombinant N-terminally His6-tagged enzyme from Escherichia coli strain C43(DE3) by nickel affinity chromatography and gel filtration Rubrivivax gelatinosus

Reaction

Reaction Comment Organism Reaction ID
magnesium-protoporphyrin IX 13-monomethyl ester + 3 NADPH + 3 H+ + 3 O2 = 3,8-divinyl protochlorophyllide a + 3 NADP+ + 5 H2O mass spectrometry identified 131-hydroxy-MgPME and 131-keto-MgPME are intermediates in the formation of the isocyclic ring, revealing the reaction chemistry that converts porphyrins to chlorins. Reaction mechanism, identification of proposed reaction intermediates in the progress of the reaction by mass spectrometry, overview Rubrivivax gelatinosus

Renatured (Commentary)

Renatured (Comment) Organism
iron reconstitution step with ferrous ammonium sulphate to increase the occupancy of the iron-binding sites, the purified single-subunit O2-dependent cyclase AcsF activity is reconstituted using a tricomponent electron transfer system consisting of NADPH, ferredoxin (Fd) and Fd:NADP+ reductase (FNR) Rubrivivax gelatinosus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
magnesium-protoporphyrin IX 13-monomethyl ester + 3 NADPH + 3 H+ + 3 O2 overall reaction Rubrivivax gelatinosus 3,8-divinyl protochlorophyllide a + 3 NADP+ + 5 H2O
-
?

Subunits

Subunits Comment Organism
dimer or trimer x * 37000, recombinant enzyme, SDS-PAGE, x * 44000, sequence calculation Rubrivivax gelatinosus
More the purified AcsF with the associated beta-DDM molecules is estimated to have a molecular mass of about 210 kDa with soluble standards, and about 180 kDa using membrane protein standards. By subtracting the contribution from detergent molecules, this indicates that AcsF is dimeric or trimeric given the predicted molecular mass of 44 kDa from the primary sequence Rubrivivax gelatinosus

Synonyms

Synonyms Comment Organism
AcsF
-
Rubrivivax gelatinosus
Mg-protoporphyrin IX monomethyl ester cyclase
-
Rubrivivax gelatinosus
MgPME cyclase
-
Rubrivivax gelatinosus
O2-dependent Mg-protoporphyrin monomethyl ester cyclase
-
Rubrivivax gelatinosus

Temperature Stability [┬░C]

Temperature Stability Minimum [┬░C] Temperature Stability Maximum [┬░C] Comment Organism
57.2
-
the melting point of AcsF Rubrivivax gelatinosus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0151
-
magnesium-protoporphyrin IX 13-monomethyl ester Michaelis-Menten kinetics, recombinant enzyme, pH 7.7, 30┬░C Rubrivivax gelatinosus
0.0177
-
magnesium-protoporphyrin IX 13-monomethyl ester sigmoidal kinetics, recombinant enzyme, pH 7.7, 30┬░C Rubrivivax gelatinosus

Cofactor

Cofactor Comment Organism Structure
Ferredoxin
-
Rubrivivax gelatinosus
NADPH
-
Rubrivivax gelatinosus

General Information

General Information Comment Organism
evolution the process for the formation of the unique isocyclic fifth ring of chlorophyll involves the conversion of Mg-protoporphyrin IX monomethyl ester (MgPME) to 3,8-divinyl protochlorophyllide a (DV PChlide a), and it requires incorporation of an oxygen atom, sourced from either water or O2, indicating the existence of two mechanistically different MgPME cyclases. Most anoxygenic phototrophic bacteria utilise an O2-sensitive radical SAM enzyme containing [4Fe-4S] and cobalamin cofactors to catalyse the reaction, while oxygenic phototrophs including cyanobacteria, algae and plants, as well as some purple bacteria, adopt an O2-dependent cyclase for the reaction. Mg-protoporphyrin IX monomethyl ester (MgPME) cyclase catalyses the formation of the isocyclic ring, the hallmark of chlorins and bacteriochlorins, producing protochlorophyllide a and contributing significantly to the absorption properties of chlorophylls and bacteriochlorophylls. Three classes of O2-dependent cyclase have been identified, all with a catalytic subunit AcsF, a putative diiron protein, but they differ in the requirement for an auxiliary subunit, either Ycf54 for the enzyme found in oxygenic phototrophs, or BciE for the alphaproteobacterial enzyme Rubrivivax gelatinosus
physiological function cyclic tetrapyrroles, are among the most abundant natural pigments on Earth. They are the major absorbers of the solar energy that drives photosynthesis, and billions of tonnes of chlorophyll are synthesised annually on land and in the oceans. The decisive biosynthetic step that determines the absorption properties of chlorophyll, and more visually its green color, is the formation of the unique isocyclic fifth ring. This process involves the conversion of Mg-protoporphyrin IX monomethyl ester (MgPME) to 3,8-divinyl protochlorophyllide a (DV PChlide a), and it requires incorporation of an oxygen atom, sourced from either water or O2, indicating the existence of two mechanistically different MgPME cyclases. Most anoxygenic phototrophic bacteria utilise an O2-sensitive radical SAM enzyme containing [4Fe-4S] and cobalamin cofactors to catalyse the reaction, while oxygenic phototrophs including cyanobacteria, algae and plants, as well as some purple bacteria, adopt an O2-dependent cyclase for the reaction. Mg-protoporphyrin IX monomethyl ester (MgPME) cyclase catalyses the formation of the isocyclic ring, the hallmark of chlorins and bacteriochlorins, producing protochlorophyllide a and contributing significantly to the absorption properties of chlorophylls and bacteriochlorophylls. The diiron cluster within AcsF is reduced by ferredoxin furnished by NADPH and ferredoxin:NADP+ reductase or by direct coupling to Photosystem I photochemistry, linking cyclase to the photosynthetic electron transport chain Rubrivivax gelatinosus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.111
-
magnesium-protoporphyrin IX 13-monomethyl ester sigmoidal kinetics, recombinant enzyme, pH 7.7, 30┬░C Rubrivivax gelatinosus
2.069
-
magnesium-protoporphyrin IX 13-monomethyl ester Michaelis-Menten kinetics, recombinant enzyme, pH 7.7, 30┬░C Rubrivivax gelatinosus