Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli BL-21 as a 6xHis tagged fusion protein | Trypanosoma brucei |
TUT4, DNA and amino acid sequence determination and analysis, expression of N-terminally His-tagged wild-type and mutant TUT4 in Escherichia coli strain BL21(DE3) | Trypanosoma brucei |
Crystallization (Comment) | Organism |
---|---|
purified recombinant N-terminally His-tagged TUT4, free or with bound 2'-deoxyribonucleoside, at a concentration of 10 mg/mL in 10 mM HEPES, pH 7.6, 70 mM KCl, 0.5 mM DTT, and in presence of 4 mM MgCl2 and 0.05 mM UTP, screening with commercial crystallization screens, vapor diffusion technique, 4°C, cryoprotection by 15% glycerol, X-ray diffraction structure determination and analysis at 2.0-2.4 A resolution | Trypanosoma brucei |
small plate-like co-crystals of purified recombinant N-terminally His-tagged TbTUT4 and UTP grow in 100 mM sodium cacodylate (pH 6.5), 200 mM calcium acetate, 18% (w/v) PEG-8000, crystal structure reveals two significantly different conformations of this TUTase:one molecule is in a relatively open apo confirmation, whereas the other displays a more compact TUTase-UTP complex | Trypanosoma brucei |
Protein Variants | Comment | Organism |
---|---|---|
D136A | inactive | Trypanosoma brucei |
D297A | Km=0.1728 (UTP), kcat=0.00033/sec (UTP) | Trypanosoma brucei |
D297N | Km=0.0139 (UTP), kcat=0.0000666/sec (UTP) | Trypanosoma brucei |
D66A | inactive | Trypanosoma brucei |
D68A | inactive | Trypanosoma brucei |
E300A | Km=0.3084 (UTP), kcat=0.0066/sec (UTP) | Trypanosoma brucei |
F52A | inactive | Trypanosoma brucei |
N147A | Km=0.1153 (UTP), kcat=0.005/sec (UTP) | Trypanosoma brucei |
R121A | Km=0.0028 (UTP), kcat=0.000033/sec (UTP),Km=0.132 (RNA), kcat= 0.005/sec (RNA) | Trypanosoma brucei |
R121F | inactive | Trypanosoma brucei |
R126F | inactive | Trypanosoma brucei |
R141A | Km=0.001 (UTP), kcat=0.00011/sec (UTP),Km=0.0726 (RNA), kcat= 0.0216/sec (RNA) | Trypanosoma brucei |
R307A | Km=0.0004 (UTP), kcat=0.000133/sec (UTP), Km=0.01 (RNA), kcat= 0.00833/sec (RNA) | Trypanosoma brucei |
S148A | Km=0.0873 (UTP), kcat=0.021/sec (UTP), Km=0.0002 (RNA), kcat= 0.0166/sec (RNA) | Trypanosoma brucei |
S188A | Km=0.034 (UTP), kcat=0.0005/sec (UTP), Km=0.0003 (RNA), kcat= 0.00166/sec (RNA) | Trypanosoma brucei |
Y189A | inactive | Trypanosoma brucei |
Y189F | Km=0.0213 (UTP), kcat=0.015/sec (UTP) | Trypanosoma brucei |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | steady-state enzyme kinetics, overview | Trypanosoma brucei | |
0.0002 | - |
RNAn | - |
Trypanosoma brucei | |
0.0002 | - |
RNAn | mutant S148A | Trypanosoma brucei | |
0.0003 | - |
RNAn | mutant S188A | Trypanosoma brucei | |
0.0004 | - |
UTP | mutant R307A | Trypanosoma brucei | |
0.001 | - |
UTP | - |
Trypanosoma brucei | |
0.001 | - |
UTP | mutant R141A | Trypanosoma brucei | |
0.0028 | - |
UTP | mutant R121A | Trypanosoma brucei | |
0.01 | - |
RNAn | mutant R307A | Trypanosoma brucei | |
0.0139 | - |
UTP | mutant D297N | Trypanosoma brucei | |
0.0213 | - |
UTP | mutant Y189F | Trypanosoma brucei | |
0.034 | - |
UTP | mutant S188A | Trypanosoma brucei | |
0.0726 | - |
RNAn | mutant R141A | Trypanosoma brucei | |
0.0873 | - |
UTP | mutant S148A | Trypanosoma brucei | |
0.1153 | - |
UTP | mutant N147A | Trypanosoma brucei | |
0.132 | - |
RNAn | mutant R121A | Trypanosoma brucei | |
0.1728 | - |
UTP | mutant D297A | Trypanosoma brucei | |
0.3084 | - |
UTP | mutant E300A | Trypanosoma brucei |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Ca2+ | influences the TUT4 conformation | Trypanosoma brucei | |
K+ | - |
Trypanosoma brucei | |
Mg2+ | maximal activity at 2 mM Mg2+ | Trypanosoma brucei | |
Mg2+ | increases the UTP substrate specificity of TUT4 and influences the enzyme conformation | Trypanosoma brucei | |
Mn2+ | replacement of magnesium with manganese leads to a dramatic stimulation of processivity and partial loss of selectivity for UTP | Trypanosoma brucei | |
additional information | divalent cations essential for catalysis | Trypanosoma brucei |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
37800 | - |
gel filtration | Trypanosoma brucei |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
UTP + RNAn | Trypanosoma brucei | - |
diphosphate + RNAn+1 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Trypanosoma brucei | Q381M1 | - |
- |
Trypanosoma brucei | Q381M1 | isozyme TUT4 | - |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant TUT4 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, ion exchange chromatography, and gel filtration | Trypanosoma brucei |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | TUT4 substrate specificity toward nucleoside triphosphate substrates with a synthetic 5'-radiolabeled 24-mer RNA as a primer, in presence of Mg2+ ions TUT4 preferentially incorporates uridylyl residues but the reaction with CTP is also detectable, TbTUT4 incorporates only one deoxynucleotide into RNA, overview. UTP binding site structure and structure-activity analysis | Trypanosoma brucei | ? | - |
? | |
UTP + RNAn | - |
Trypanosoma brucei | diphosphate + RNAn+1 | - |
? | |
UTP + RNAn | a template-independent RNA nucleotidyltransferases that specifically recognize UTP, it possesses conserved catalytic and UTP recognition domains. A single nucleoside triphosphate is bound in the active site by a complex network of interactions between amino acid residues, a magnesium ion and highly ordered water molecules with the UTPs base, ribose and phosphate moieties, structure-function analysis, overview | Trypanosoma brucei | diphosphate + RNAn+1 | - |
? |
Subunits | Comment | Organism |
---|---|---|
? | x * 37800, TUT4, crystal structure analysis | Trypanosoma brucei |
More | TbTUT4 represents a minimal catalytically active RNA uridylyltransferase consisting of only two domains that define the catalytic center at the bottom of the nucleoside triphosphate and RNA substrate binding cleft. The enzyme shows two significantly different conformations: one molecule is in a relatively open apo conformation, whereas the other displays a more compact TUTase-UTP complex, structure-function analysis, overview | Trypanosoma brucei |
Synonyms | Comment | Organism |
---|---|---|
More | the enzyme belongs to a large enzyme superfamily typified by DNA polymerase beta | Trypanosoma brucei |
TbTUT4 | - |
Trypanosoma brucei |
terminal uridylyltransferase | - |
Trypanosoma brucei |
TUT4 | - |
Trypanosoma brucei |
TUTase | - |
Trypanosoma brucei |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
27 | - |
assay at | Trypanosoma brucei |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.000033 | - |
UTP | mutant R121A | Trypanosoma brucei | |
0.0000666 | - |
UTP | mutant D297N | Trypanosoma brucei | |
0.00011 | - |
UTP | mutant R141A | Trypanosoma brucei | |
0.000133 | - |
UTP | mutant R307A | Trypanosoma brucei | |
0.00033 | - |
UTP | mutant D297A | Trypanosoma brucei | |
0.0005 | - |
UTP | mutant S188A | Trypanosoma brucei | |
0.00166 | - |
RNA | mutant S188A | Trypanosoma brucei | |
0.005 | - |
UTP | mutant N147A | Trypanosoma brucei | |
0.005 | - |
RNA | mutant R121A | Trypanosoma brucei | |
0.0066 | - |
UTP | mutant E300A | Trypanosoma brucei | |
0.00833 | - |
RNA | mutant R307A | Trypanosoma brucei | |
0.015 | - |
UTP | mutant Y189F | Trypanosoma brucei | |
0.0166 | - |
RNA | mutant S148A | Trypanosoma brucei | |
0.021 | - |
UTP | mutant S148A | Trypanosoma brucei | |
0.0216 | - |
RNA | mutant R141A | Trypanosoma brucei |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Trypanosoma brucei |