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Literature summary extracted from

  • Stagno, J.; Aphasizheva, I.; Rosengarth, A.; Luecke, H.; Aphasizhev, R.
    UTP-bound and Apo structures of a minimal RNA uridylyltransferase (2007), J. Mol. Biol., 366, 882-899.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.7.52 expressed in Escherichia coli BL-21 as a 6xHis tagged fusion protein Trypanosoma brucei
2.7.7.52 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 (Commentary)

EC Number Crystallization (Comment) Organism
2.7.7.52 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
2.7.7.52 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

EC Number Protein Variants Comment Organism
2.7.7.52 D136A inactive Trypanosoma brucei
2.7.7.52 D297A Km=0.1728 (UTP), kcat=0.00033/sec (UTP) Trypanosoma brucei
2.7.7.52 D297N Km=0.0139 (UTP), kcat=0.0000666/sec (UTP) Trypanosoma brucei
2.7.7.52 D66A inactive Trypanosoma brucei
2.7.7.52 D68A inactive Trypanosoma brucei
2.7.7.52 E300A Km=0.3084 (UTP), kcat=0.0066/sec (UTP) Trypanosoma brucei
2.7.7.52 F52A inactive Trypanosoma brucei
2.7.7.52 N147A Km=0.1153 (UTP), kcat=0.005/sec (UTP) Trypanosoma brucei
2.7.7.52 R121A Km=0.0028 (UTP), kcat=0.000033/sec (UTP),Km=0.132 (RNA), kcat= 0.005/sec (RNA) Trypanosoma brucei
2.7.7.52 R121F inactive Trypanosoma brucei
2.7.7.52 R126F inactive Trypanosoma brucei
2.7.7.52 R141A Km=0.001 (UTP), kcat=0.00011/sec (UTP),Km=0.0726 (RNA), kcat= 0.0216/sec (RNA) Trypanosoma brucei
2.7.7.52 R307A Km=0.0004 (UTP), kcat=0.000133/sec (UTP), Km=0.01 (RNA), kcat= 0.00833/sec (RNA) Trypanosoma brucei
2.7.7.52 S148A Km=0.0873 (UTP), kcat=0.021/sec (UTP), Km=0.0002 (RNA), kcat= 0.0166/sec (RNA) Trypanosoma brucei
2.7.7.52 S188A Km=0.034 (UTP), kcat=0.0005/sec (UTP), Km=0.0003 (RNA), kcat= 0.00166/sec (RNA) Trypanosoma brucei
2.7.7.52 Y189A inactive Trypanosoma brucei
2.7.7.52 Y189F Km=0.0213 (UTP), kcat=0.015/sec (UTP) Trypanosoma brucei

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.7.7.52 additional information
-
 additional information steady-state enzyme kinetics, overview Trypanosoma brucei
2.7.7.52 0.0002
-
 RNAn
-
 Trypanosoma brucei
2.7.7.52 0.0002
-
 RNAn mutant S148A Trypanosoma brucei
2.7.7.52 0.0003
-
 RNAn mutant S188A Trypanosoma brucei
2.7.7.52 0.0004
-
 UTP mutant R307A Trypanosoma brucei
2.7.7.52 0.001
-
 UTP
-
 Trypanosoma brucei
2.7.7.52 0.001
-
 UTP mutant R141A Trypanosoma brucei
2.7.7.52 0.0028
-
 UTP mutant R121A Trypanosoma brucei
2.7.7.52 0.01
-
 RNAn mutant R307A Trypanosoma brucei
2.7.7.52 0.0139
-
 UTP mutant D297N Trypanosoma brucei
2.7.7.52 0.0213
-
 UTP mutant Y189F Trypanosoma brucei
2.7.7.52 0.034
-
 UTP mutant S188A Trypanosoma brucei
2.7.7.52 0.0726
-
 RNAn mutant R141A Trypanosoma brucei
2.7.7.52 0.0873
-
 UTP mutant S148A Trypanosoma brucei
2.7.7.52 0.1153
-
 UTP mutant N147A Trypanosoma brucei
2.7.7.52 0.132
-
 RNAn mutant R121A Trypanosoma brucei
2.7.7.52 0.1728
-
 UTP mutant D297A Trypanosoma brucei
2.7.7.52 0.3084
-
 UTP mutant E300A Trypanosoma brucei

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.7.7.52 Ca2+ influences the TUT4 conformation Trypanosoma brucei
2.7.7.52 K+
-
 Trypanosoma brucei
2.7.7.52 Mg2+ maximal activity at 2 mM Mg2+ Trypanosoma brucei
2.7.7.52 Mg2+ increases the UTP substrate specificity of TUT4 and influences the enzyme conformation Trypanosoma brucei
2.7.7.52 Mn2+ replacement of magnesium with manganese leads to a dramatic stimulation of processivity and partial loss of selectivity for UTP Trypanosoma brucei
2.7.7.52 additional information divalent cations essential for catalysis Trypanosoma brucei

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.7.7.52 37800
-
 gel filtration Trypanosoma brucei

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.7.7.52 UTP + RNAn Trypanosoma brucei
-
 diphosphate + RNAn+1
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.7.7.52 Trypanosoma brucei Q381M1
-
-
2.7.7.52 Trypanosoma brucei Q381M1 isozyme TUT4
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.7.52 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 and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.7.52 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 ?
-
 ?
2.7.7.52 UTP + RNAn
-
 Trypanosoma brucei diphosphate + RNAn+1
-
 ?
2.7.7.52 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 UTP’s base, ribose and phosphate moieties, structure-function analysis, overview Trypanosoma brucei diphosphate + RNAn+1
-
 ?

Subunits

EC Number Subunits Comment Organism
2.7.7.52 ? x * 37800, TUT4, crystal structure analysis Trypanosoma brucei
2.7.7.52 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

EC Number Synonyms Comment Organism
2.7.7.52 More the enzyme belongs to a large enzyme superfamily typified by DNA polymerase beta Trypanosoma brucei
2.7.7.52 TbTUT4
-
 Trypanosoma brucei
2.7.7.52 terminal uridylyltransferase
-
 Trypanosoma brucei
2.7.7.52 TUT4
-
 Trypanosoma brucei
2.7.7.52 TUTase
-
 Trypanosoma brucei

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.7.7.52 27
-
 assay at Trypanosoma brucei

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.7.7.52 0.000033
-
 UTP mutant R121A Trypanosoma brucei
2.7.7.52 0.0000666
-
 UTP mutant D297N Trypanosoma brucei
2.7.7.52 0.00011
-
 UTP mutant R141A Trypanosoma brucei
2.7.7.52 0.000133
-
 UTP mutant R307A Trypanosoma brucei
2.7.7.52 0.00033
-
 UTP mutant D297A Trypanosoma brucei
2.7.7.52 0.0005
-
 UTP mutant S188A Trypanosoma brucei
2.7.7.52 0.00166
-
 RNA mutant S188A Trypanosoma brucei
2.7.7.52 0.005
-
 UTP mutant N147A Trypanosoma brucei
2.7.7.52 0.005
-
 RNA mutant R121A Trypanosoma brucei
2.7.7.52 0.0066
-
 UTP mutant E300A Trypanosoma brucei
2.7.7.52 0.00833
-
 RNA mutant R307A Trypanosoma brucei
2.7.7.52 0.015
-
 UTP mutant Y189F Trypanosoma brucei
2.7.7.52 0.0166
-
 RNA mutant S148A Trypanosoma brucei
2.7.7.52 0.021
-
 UTP mutant S148A Trypanosoma brucei
2.7.7.52 0.0216
-
 RNA mutant R141A Trypanosoma brucei

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.7.7.52 8
-
 assay at Trypanosoma brucei