Functional importance of motif I of pseudouridine synthases: mutagenesis of aligned lysine and proline residues

Spedaliere, C.J.; Hamilton, C.S.; Mueller, E.G.; Biochemistry 39, 9459-9465 (2000)

 show all sequences of 5.4.99.25

Data extracted from this reference:

Engineering
Amino acid exchange Commentary Organism
K19M kcat is 8fold lower than wild-type value. KM-value is 11fold higher than wild-type value Escherichia coli
K19R kcat is 10fold lower than wild-type value. KM-value is 6fold higher than wild-type value Escherichia coli
additional information mutant enzymes with substitution of the nearly invariant lysine and proline residues in motif I of RluA display only very mild kinetic impairment. Substitution of the aligned lysine and proline residues reduces structural stability Escherichia coli
P20G kcat is 4.8fold lower than wild-type value. KM-value is 2.2fold higher than wild-type value Escherichia coli
P20L kcat is 2.1fold lower than wild-type value. KM-value is 3.5fold higher than wild-type value Escherichia coli
KM Value [mM]
KM Value [mM] KM Value Maximum [mM] Substrate Commentary Organism Structure
0.000148
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, wild-type enzyme Escherichia coli
-
0.000324
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme P20G Escherichia coli
-
0.000517
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme P20L Escherichia coli
-
0.0009
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme K19R Escherichia coli
-
0.00161
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme K19M Escherichia coli
-
Organism
Organism Primary Accession No. (UniProt) Commentary Textmining
Escherichia coli P60340
-
-
Substrates and Products (Substrate)
Substrates Commentary Substrates Literature (Substrates) Organism Products Commentary (Products) Literature (Products) Organism (Products) Reversibility
tRNA uridine55 substrate: Escherichia coli tRNAPhe 702203 Escherichia coli tRNA pseudouridine552
-
-
-
 ?
Temperature Optimum [°C]
Temperature Optimum [°C] Temperature Optimum Maximum [°C] Commentary Organism
37
-
 assay at Escherichia coli
Temperature Stability [°C]
Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Commentary Organism
36
-
 Tm-value for mutant enzyme P20L is 35.9°C Escherichia coli
38
-
 Tm-value for mutant enzyme P20G is 38.3°C Escherichia coli
40
-
 Tm-value for wild-type enzyme is 40.2°C Escherichia coli
Turnover Number [1/s]
Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Commentary Organism Structure
0.011
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme K19R Escherichia coli
-
0.012
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme K19M Escherichia coli
-
0.11
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, wild-type enzyme Escherichia coli
-
0.23
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme P20G Escherichia coli
-
0.52
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme P20L Escherichia coli
-
pH Optimum
pH Optimum Minimum pH Optimum Maximum Commentary Organism
7.5
-
 assay at Escherichia coli
Engineering (protein specific)
Amino acid exchange Commentary Organism
K19M kcat is 8fold lower than wild-type value. KM-value is 11fold higher than wild-type value Escherichia coli
K19R kcat is 10fold lower than wild-type value. KM-value is 6fold higher than wild-type value Escherichia coli
additional information mutant enzymes with substitution of the nearly invariant lysine and proline residues in motif I of RluA display only very mild kinetic impairment. Substitution of the aligned lysine and proline residues reduces structural stability Escherichia coli
P20G kcat is 4.8fold lower than wild-type value. KM-value is 2.2fold higher than wild-type value Escherichia coli
P20L kcat is 2.1fold lower than wild-type value. KM-value is 3.5fold higher than wild-type value Escherichia coli
KM Value [mM] (protein specific)
KM Value [mM] KM Value Maximum [mM] Substrate Commentary Organism Structure
0.000148
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, wild-type enzyme Escherichia coli
-
0.000324
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme P20G Escherichia coli
-
0.000517
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme P20L Escherichia coli
-
0.0009
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme K19R Escherichia coli
-
0.00161
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme K19M Escherichia coli
-
Substrates and Products (Substrate) (protein specific)
Substrates Commentary Substrates Literature (Substrates) Organism Products Commentary (Products) Literature (Products) Organism (Products) Reversibility
tRNA uridine55 substrate: Escherichia coli tRNAPhe 702203 Escherichia coli tRNA pseudouridine552
-
-
-
 ?
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C] Temperature Optimum Maximum [°C] Commentary Organism
37
-
 assay at Escherichia coli
Temperature Stability [°C] (protein specific)
Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Commentary Organism
36
-
 Tm-value for mutant enzyme P20L is 35.9°C Escherichia coli
38
-
 Tm-value for mutant enzyme P20G is 38.3°C Escherichia coli
40
-
 Tm-value for wild-type enzyme is 40.2°C Escherichia coli
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Commentary Organism Structure
0.011
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme K19R Escherichia coli
-
0.012
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme K19M Escherichia coli
-
0.11
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, wild-type enzyme Escherichia coli
-
0.23
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme P20G Escherichia coli
-
0.52
-
 tRNA uridine55 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme P20L Escherichia coli
-
pH Optimum (protein specific)
pH Optimum Minimum pH Optimum Maximum Commentary Organism
7.5
-
 assay at Escherichia coli
KCat/KM [mM/s]
kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Commentary Organism Structure
7.43
-
 tRNA uridine32 pH 7.5, 37°C, Escherichia coli tRNAPhe, wild-type enzyme Escherichia coli
-
7.45
-
 tRNA uridine32 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme K19M Escherichia coli
-
12.2
-
 tRNA uridine32 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme K19R Escherichia coli
-
709.8
-
 tRNA uridine32 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme P20G Escherichia coli
-
1006
-
 tRNA uridine32 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme P20L Escherichia coli
-
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1] KCat/KM Value Maximum [1/mMs-1] Substrate Commentary Organism Structure
7.43
-
 tRNA uridine32 pH 7.5, 37°C, Escherichia coli tRNAPhe, wild-type enzyme Escherichia coli
-
7.45
-
 tRNA uridine32 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme K19M Escherichia coli
-
12.2
-
 tRNA uridine32 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme K19R Escherichia coli
-
709.8
-
 tRNA uridine32 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme P20G Escherichia coli
-
1006
-
 tRNA uridine32 pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme P20L Escherichia coli
-

See also following references to EC number 5.4.99.25 (sorted by year of publication):
No.1st authorPub
Med		titleorganimsjournalvolumepagesyearActivating CompoundApplicationCloned(Commentary)Crystallization (Commentary)EngineeringGeneral StabilityInhibitorsKM Value [mM]LocalizationMetals/IonsMolecular Weight [Da]Natural Substrates/ Products (Substrates)Organic Solvent StabilityOrganismOxidation StabilityPosttranslational ModificationPurification (Commentary)ReactionRenatured (Commentary)Source TissueSpecific Activity [micromol/min/mg]Storage StabilitySubstrates and Products (Substrate)SubunitsTemperature Optimum [°C]Temperature Range [°C]Temperature Stability [°C]Turnover Number [1/s]pH OptimumpH RangepH StabilityCofactorKi Value [mM]pI ValueIC50 ValueActivating Compound (protein specific)Application (protein specific)Cloned(Commentary) (protein specific)Cofactor (protein specific)Crystallization (Commentary) (protein specific)Engineering (protein specific)General Stability (protein specific)IC50 Value (protein specific)Inhibitors (protein specific)Ki Value [mM] (protein specific)KM Value [mM] (protein specific)Localization (protein specific)Metals/Ions (protein specific)Molecular Weight [Da] (protein specific)Natural Substrates/ Products (Substrates) (protein specific)Organic Solvent Stability (protein specific)Oxidation Stability (protein specific)Posttranslational Modification (protein specific)Purification (Commentary) (protein specific)Renatured (Commentary) (protein specific)Source Tissue (protein specific)Specific Activity [micromol/min/mg] (protein specific)Storage Stability (protein specific)Substrates and Products (Substrate) (protein specific)Subunits (protein specific)Temperature Optimum [°C] (protein specific)Temperature Range [°C] (protein specific)Temperature Stability [°C] (protein specific)Turnover Number [1/s] (protein specific)pH Optimum (protein specific)pH Range (protein specific)pH Stability (protein specific)pI Value (protein specific)ExpressionGeneral InformationGeneral Information (protein specific)Expression (protein specific)KCat/KM [mM/s]KCat/KM [mM/s] (protein specific)
695108GurhaArchaeal Pus10 proteins can pr ...Methanocaldococcus jannaschii, Pyrococcus furiosusRNA142521-25272008--2----------6--------2--------------2--------------------2---------------
705960MullerIdentification of determinants ...Pyrococcus abyssiNucleic Acids Res.355610-562420071---2--------2--------1------------1----2-----------------1---------------
706710GurhaDifferential roles of archaeal ...Methanocaldococcus jannaschiiRNA Biol.4101-10920072-1----------4---1----1------------2-1--------------------1---------------
705958RooversFormation of the conserved pse ...Pyrococcus furiosusNucleic Acids Res.344293-430120061-1----------1--1-----1-11---------1-1---------------1----1-11-------11---
661179PhannachetDissecting the roles of a stri ...Thermotoga maritimaBiochemistry4415488-154942005---12--1-----4---1----2----1-----------12----1------------2----1----------
663343HoangPrecursor complex structure of ...Escherichia coliProtein Sci.142201-22062005---11--1-----2---1----1----1-----------11----1------------1----1----------
652542ChaudhuriCrystal structure of the apo f ...Mycobacterium tuberculosisJ. Biol. Chem.27924585-245912004---1---------1--1----------------------1-------------1--------------------
706711SpedaliereNot all pseudouridine synthase ...Escherichia coliRNA10192-1992004------1------3--------1--------------------1--------------1---------------
653587PanStructure of tRNA pseudouridin ...Escherichia coli, Thermotoga maritimaProc. Natl. Acad. Sci. USA10012648-126532003---2---------5--22---------------------2-------------2--------------------
704264UrbonaviciusThree modifications in the D a ...Escherichia coli, Shigella flexneriJ. Bacteriol.1845348-53572002-------------2-------------------------------------------------------55---
705550KinghornPhysiological analysis of the ...Escherichia coliMicrobiology1483511-35202002--1----------1-----------------------1-------------------------------22---
650781HoangCocrystal structure of a tRNA ...Escherichia coliCell107929-9392001---1---------1---1---------------------1----------------------------------
702203SpedaliereFunctional importance of motif ...Escherichia coliBiochemistry399459-94652000----5--5-----1--------1-1-351-----------5----5------------1-1-351-------55
706733GutgsellDeletion of the Escherichia co ...Escherichia coliRNA61870-18812000-------------1-------------------------------------------------------11---
648867RamamurthyRole of cysteine residues in p ...Escherichia coliBiochemistry3813106-131111999--1-5--5-----1--11----1-1--51--------1--5----5-------1----1-1--51-------55
701450PienkowskaA cell-free yellow lupin extra ...Lupinus luteusActa Biochim. Pol.45745-7541998-------------1-----1--1--------------------------------1--1---------------
702196GuMolecular recognition of tRNA ...Escherichia coliBiochemistry37339-3431998-------2-----2--------1-1--21----------------2------------1-1--21---------
648862BeckerThe yeast gene YNL292w encodes ...Saccharomyces cerevisiaeNucleic Acids Res.254493-44991997--1-----2--1-4--1-----2-1---1--------1--------2--1---1----2-1---1----11---
706713NursePurification, cloning, and pro ...Escherichia coliRNA1102-1121995--1----------5--1-----11-------------1---------------1----11--------------