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Literature summary for 2.7.7.65 extracted from

  • Lengalova, A.; Fojtikova-Proskova, V.; Vavra, J.; Martinek, V.; Stranava, M.; Shimizu, T.; Martinkova, M.
    Kinetic analysis of a globin-coupled diguanylate cyclase, YddV effects of heme iron redox state, axial ligands, and heme distal mutations on catalysis (2019), J. Inorg. Biochem., 201, 110833 .
    View publication on PubMed
Search for more literature for 2.7.7.65

Cloned(Commentary)

Cloned (Comment) Organism
gene dosC or yddV, stable expression of MBP-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Escherichia coli

Protein Variants

Protein Variants Comment Organism
H93A site-directed mutagenesis, the mutant has no heme and is inactive Escherichia coli
H98A site-directed mutagenesis Escherichia coli
L65G site-directed mutagenesis, heme distal amino acid replacement leading to reduced activity compared to wild-type Escherichia coli
L65M site-directed mutagenesis, heme distal amino acid replacement Escherichia coli
L65Q site-directed mutagenesis, heme distal amino acid replacement leading to reduced activity compared to wild-type Escherichia coli
L65T site-directed mutagenesis, heme distal amino acid replacement Escherichia coli
R365A site-directed mutagenesis Escherichia coli
Y43A site-directed mutagenesis, heme distal amino acid replacement leading to reduced activity compared to wild-type Escherichia coli
Y43F site-directed mutagenesis, heme distal amino acid replacement Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information Michaelis-Menten kinetics, kinetic analysis Escherichia coli
0.022
-
 GTP enzyme with 5-coordinate Fe(III) heme mutant L65G, pH 8.0, 24°C Escherichia coli
0.029
-
 GTP enzyme with 5-coordinate Fe(III) heme mutant Y43F, pH 8.0, 24°C Escherichia coli
0.042
-
 GTP enzyme with 5-coordinate Fe(III) heme mutant L65M, pH 8.0, 24°C Escherichia coli
0.054
-
 GTP enzyme with 5-coordinate Fe(III) heme mutant Y43A, pH 8.0, 24°C Escherichia coli
0.063
-
 GTP enzyme with 5-coordinate Fe(III) heme mutant L65T, pH 8.0, 24°C Escherichia coli
0.084
-
 GTP wild-type enzyme with 5-coordinate Fe(III) heme, pH 8.0, 24°C Escherichia coli
0.101
-
 GTP enzyme with 5-coordinate Fe(III) heme mutant L65Q, pH 8.0, 24°C Escherichia coli

Metals/Ions

Metals/Ions Comment Organism Structure
Fe2+ the most active form of MBP-tagged YddV has an 5-coordinate Fe(III) heme complex Escherichia coli
Mg2+ catalysis requires either Mg2+ or Mn2+ Escherichia coli
Mn2+ catalysis requires either Mg2+ or Mn2+ Escherichia coli
additional information catalysis requires either Mg2+ or Mn2+, other divalent metal ions (Ca2+, Co2+, Ni2+, Zn2+, and Cd2+) give no activity Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2 GTP Escherichia coli
-
 2 diphosphate + cyclic di-3',5'-guanylate
-
 ?

Organism

Organism UniProt Comment Textmining
Escherichia coli P0AA89
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant MBP-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by amylose affinity chromatography, gel filtration, and ultrafiltration Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2 GTP
-
 Escherichia coli 2 diphosphate + cyclic di-3',5'-guanylate
-
 ?

Subunits

Subunits Comment Organism
dimer MBP-tagged YddV is primarily dimeric in solution Escherichia coli
More the inactive heme-free H93A mutant is primarily octameric, suggesting that catalytically active dimer formation requires heme binding. The dimer to tetramer ratios for MBP-tagged YddV in the presence and absence of GTP (and therefore of c-di-GMP) are 3.7:1 and 4.3:1, respectively. Like the Fe(III) form, the Fe(II)-O2, Fe(III)-CN- and Fe(III)-imidazole forms of YddV-MBP exist primarily as dimers, with tetramers being minor components Escherichia coli

Synonyms

Synonyms Comment Organism
YddV
-
 Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
24
-
 assay at Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0025
-
 GTP enzyme with 5-coordinate Fe(III) heme mutant L65G, pH 8.0, 24°C Escherichia coli
0.0043
-
 GTP enzyme with 5-coordinate Fe(III) heme mutant L65Q, pH 8.0, 24°C Escherichia coli
0.005
-
 GTP enzyme with mutant 5-coordinate Fe(II) heme, pH 8.0, 24°C Escherichia coli
0.005
-
 GTP enzyme with mutant 5-coordinate Fe(II)-CO heme, pH 8.0, 24°C Escherichia coli
0.009
-
 GTP enzyme with 5-coordinate Fe(III) heme mutant Y43A, pH 8.0, 24°C Escherichia coli
0.0115
-
 GTP enzyme with 5-coordinate Fe(III) heme mutant Y43F, pH 8.0, 24°C Escherichia coli
0.015
-
 GTP enzyme with 5-coordinate Fe(III) heme mutant L65M, pH 8.0, 24°C Escherichia coli
0.016
-
 GTP enzyme with mutant 5-coordinate Fe(II)-O2 heme, pH 8.0, 24°C Escherichia coli
0.02
-
 GTP enzyme with 5-coordinate Fe(III) heme, pH 8.0, 24°C Escherichia coli
0.078
-
 GTP enzyme with 5-coordinate Fe(III) heme mutant L65T, pH 8.0, 24°C Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8.5 9
-
 Escherichia coli

pH Range

pH Minimum pH Maximum Comment Organism
6 10 the enzyme is inactive below pH 6.0 and above pH 10.0, optimal pH at pH 8.5-9.00, 50% of maximal activity at pH 7.0, 80% of maximal activity at pH 8.0 Escherichia coli

Cofactor

Cofactor Comment Organism Structure
heme the most active form of MBP-tagged YddV has an 5-coordinate Fe(III) heme complex. Heme distal residues play key regulatory roles by mediating signal transduction between the sensing and functional domains Escherichia coli

General Information

General Information Comment Organism
malfunction the inactive heme-free H93A mutant is primarily octameric, suggesting that catalytically active dimer formation requires heme binding Escherichia coli
physiological function globin-coupled oxygen sensor with diguanylate cyclase activity from Escherichia coli, regulates cyclic-di-GMP synthesis based on oxygen availability. Cyclic-di-guanosine-5'-monophosphate (c-di-GMP) is an important bacterial second messenger that regulates many key physiological functions including cell motility, differentiation, development, virulence, biofilm formation, cell-cell communication, and environmental persistence Escherichia coli

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.043
-
 GTP enzyme with 5-coordinate Fe(III) heme mutant L65Q, pH 8.0, 24°C Escherichia coli
0.114
-
 GTP enzyme with 5-coordinate Fe(III) heme mutant L65G, pH 8.0, 24°C Escherichia coli
0.167
-
 GTP enzyme with 5-coordinate Fe(III) heme mutant Y43A, pH 8.0, 24°C Escherichia coli
0.238
-
 GTP enzyme with 5-coordinate Fe(III) heme, pH 8.0, 24°C Escherichia coli
0.357
-
 GTP enzyme with 5-coordinate Fe(III) heme mutant L65M, pH 8.0, 24°C Escherichia coli
0.397
-
 GTP enzyme with 5-coordinate Fe(III) heme mutant Y43F, pH 8.0, 24°C Escherichia coli
1.238
-
 GTP enzyme with 5-coordinate Fe(III) heme mutant L65T, pH 8.0, 24°C Escherichia coli