Cloned (Comment) | Organism |
---|---|
recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 gold | Pseudomonas putida |
Crystallization (Comment) | Organism |
---|---|
RifI2 X-ray diffraction structure determination and analysis at 2.15 A resolution, modelling | Pseudomonas putida |
Protein Variants | Comment | Organism |
---|---|---|
N193D | site-directed mutagenesis, inactive mutant | Pseudomonas putida |
N193L | site-directed mutagenesis, inactive mutant | Pseudomonas putida |
N193Q | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Pseudomonas putida |
N193S | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Pseudomonas putida |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.43 | - |
NAD+ | pH 8.8, 25°C, with quinate | Pseudomonas putida | |
0.71 | - |
NAD+ | pH 8.8, 25°C, with shikimate | Pseudomonas putida | |
1.88 | - |
L-quinate | pH 8.8, 25°C, with NAD+ | Pseudomonas putida | |
2.18 | - |
shikimate | pH 8.8, 25°C, with NADP+ | Pseudomonas putida | |
2.65 | - |
NADP+ | pH 8.8, 25°C, with shikimate | Pseudomonas putida | |
3.38 | - |
shikimate | pH 8.8, 25°C, with NAD+ | Pseudomonas putida |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
additional information | divalent cations are not required for the activity of RifI2, as the reaction rate is not altered in the presence of 5 mM Ca2+, Mg2+, Mn2+, or Zn2+. Similarly, the reaction is not affected by the addition of 10 mM EDTA to chelate any endogenous divalent cations associated with the enzyme | Pseudomonas putida |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-quinate + NAD+ | Pseudomonas putida | - |
3-dehydroquinate + NADH + H+ | - |
? | |
L-quinate + NAD+ | Pseudomonas putida KT 2240 | - |
3-dehydroquinate + NADH + H+ | - |
? | |
shikimate + NAD+ | Pseudomonas putida | - |
3-dehydroshikimate + NADH + H+ | - |
? | |
shikimate + NAD+ | Pseudomonas putida KT 2240 | - |
3-dehydroshikimate + NADH + H+ | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas putida | Q88JP1 | - |
- |
Pseudomonas putida KT 2240 | Q88JP1 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 gold by nickel affinity chromatography, dialysis, tag cleavage through TEV protease and removal by nickel affinity chromatography, followed by gel filtration | Pseudomonas putida |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-quinate + NAD+ | - |
Pseudomonas putida | 3-dehydroquinate + NADH + H+ | - |
? | |
L-quinate + NAD+ | - |
Pseudomonas putida KT 2240 | 3-dehydroquinate + NADH + H+ | - |
? | |
shikimate + NAD+ | - |
Pseudomonas putida | 3-dehydroshikimate + NADH + H+ | - |
? | |
shikimate + NAD+ | - |
Pseudomonas putida KT 2240 | 3-dehydroshikimate + NADH + H+ | - |
? | |
shikimate + NADP+ | very low activity with NADP+ | Pseudomonas putida | 3-dehydroshikimate + NADPH + H+ | - |
? | |
shikimate + NADP+ | very low activity with NADP+ | Pseudomonas putida KT 2240 | 3-dehydroshikimate + NADPH + H+ | - |
? |
Subunits | Comment | Organism |
---|---|---|
dimer | distinct mode of dimerization in which the individual molecules interact in a back-to-front manner, overview. The molecules of the RifI2 dimer associate via hydrophobic interactions between residues on alpha-helix alpha8 of the first molecule and alpha10' of the second molecule. In addition, the side chain of Gln167 on apha-helix alpha8 forms a hydrogen bond with the guanidinium group of Arg243' on beta-strand beta10'. A second hydrogen bond to Arg243' may be made by Asn171. Asp53 and Arg56 on alpha-helix alpha2 bind the imidazole ring of His31' on alpha-helix alpha1' | Pseudomonas putida |
Synonyms | Comment | Organism |
---|---|---|
RifI2 | - |
Pseudomonas putida |
SDH/QDH | - |
Pseudomonas putida |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0055 | - |
NAD+ | pH 8.8, 25°C, with quinate | Pseudomonas putida | |
0.0063 | - |
L-quinate | pH 8.8, 25°C, with NAD+ | Pseudomonas putida | |
0.022 | - |
NADP+ | pH 8.8, 25°C, with shikimate | Pseudomonas putida | |
0.027 | - |
shikimate | pH 8.8, 25°C, with NADP+ | Pseudomonas putida | |
2.8 | - |
NAD+ | pH 8.8, 25°C, with shikimate | Pseudomonas putida | |
3.9 | - |
shikimate | pH 8.8, 25°C, with NAD+ | Pseudomonas putida |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
additional information | in the RifI2 structure, the NADP+-specifying motif is replaced with Asp-Pro-Ser-Thr-Ala-Arg (residues 156-161). The side chain of Asp156 binds to the adenine ribose of NAD+, while its negative charge is predicted to repel the additional phosphate of NADP+. RifI2 possesses low apparent binding affinity for NADP+. Analysis of the cofactor-binding sites of the RifI2NAD+ complex and structure comparison, inportance of the invariant residue Asn193 in the cofactor-binding site, overview | Pseudomonas putida | |
NAD+ | the C-terminal domain of each protomer in the RifI2 asymmetric unit contains a bound molecule of NAD+, Cofactor binding structure and mechanism, detailed overview | Pseudomonas putida | |
NADH | - |
Pseudomonas putida |
General Information | Comment | Organism |
---|---|---|
additional information | enzyme RifI2 lacks a conserved C-terminal alpha-helix | Pseudomonas putida |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0034 | - |
L-quinate | pH 8.8, 25°C, with NAD+ | Pseudomonas putida | |
0.0083 | - |
NADP+ | pH 8.8, 25°C, with shikimate | Pseudomonas putida | |
0.012 | - |
shikimate | pH 8.8, 25°C, with NADP+ | Pseudomonas putida | |
0.013 | - |
NAD+ | pH 8.8, 25°C, with quinate | Pseudomonas putida | |
1.15 | - |
shikimate | pH 8.8, 25°C, with NAD+ | Pseudomonas putida | |
3.95 | - |
NAD+ | pH 8.8, 25°C, with shikimate | Pseudomonas putida |