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Literature summary extracted from

  • Petrella, S.; Capton, E.; Raynal, B.; Giffard, C.; Thureau, A.; Bonnete, F.; Alzari, P.M.; Aubry, A.; Mayer, C.
    Overall structures of Mycobacterium tuberculosis DNA gyrase reveal the role of a Corynebacteriales GyrB-specific insert in ATPase activity (2019), Structure, 27, 579-589.e5 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
5.6.2.2 Mycobacterium tuberculosis encodes only one type II topoisomerase, i.e. DNA gyrase, recombinant expression of wild-type and mutant enzymes Mycobacterium tuberculosis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
5.6.2.2 recombinant engineered DNA gyrase GyrBA57 in apo- and AMPPNP-bound forms, sitting-drop vapour diffusion method, mixing of 10 mg/mlGyrBA57 in presence or absence of Mg2+ or 5 mM MgCl2 plus 2.5 mM AMPPNP, or in the presence of 1 mM AMPPNP plus 8 mM oligonucleotides, with 100 mM sodium acetate, 100 mM MES, pH 6.5, 26% PEG 400, 25% ethylene glycol, and 10 mM MgCl2 (for Apo-GyrBA57 and AMPPNP-bound GyrBA57, respectively), X-ray diffraction structure determination and analysis at 2.6 A and 3.3 A resolution, respectively Mycobacterium tuberculosis

Protein Variants

EC Number Protein Variants Comment Organism
5.6.2.2 D211K/E212K/E213K/E214K site-directed mutagenesis, the GyrBAKKKK mutant comprises the charge-reversal mutation of all the acidic residues composing the DEEE loop into basic ones (211DEEE214 into 211KKKK214) Mycobacterium tuberculosis
5.6.2.2 E221K/D225K/E228K/R236E/H244A site-directed mutagenesis, mutant GyrBAmC-loop, the substitution of five residues in the C-loop which are implicated in salt bridges or strong hydrogen interactions participating to the stabilization of the N-gate extremely open conformation Mycobacterium tuberculosis
5.6.2.2 additional information the subunits GyrB and GyrA57 are linked into a single polypeptide chain (termed GyrBA57) leading to an enzyme (full-length GyrB-GyrA57)2 with a total molecular weight of almost 260 kDa (by analytical ultracentrifugation) and four ATPases domains. Construction of the GyrBA57DELTAC-loop truncated mutant. The two GyrBA57 and GyrBA57DC-loop enzymes are dimeric in solution. Structure modelling Mycobacterium tuberculosis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
5.6.2.2 0.3
-
 ATP enzyme mutant GyrBADELTAC-loop, pH 7.5, 30°C Mycobacterium tuberculosis
5.6.2.2 0.7
-
 ATP enzyme mutant GyrBAmC-loop (E221K/D225K/E228K/R236E/H244A), pH 7.5, 30°C Mycobacterium tuberculosis
5.6.2.2 0.7
-
 ATP wild-type enzyme GyrBA, pH 7.5, 30°C Mycobacterium tuberculosis
5.6.2.2 0.75
-
 ATP enzyme dimeric mutant GyrBA57DELTAC-loop, pH 7.5, 30°C Mycobacterium tuberculosis
5.6.2.2 0.8
-
 ATP enzyme dimeric mutant GyrBA57, pH 7.5, 30°C Mycobacterium tuberculosis
5.6.2.2 1
-
 ATP enzyme mutant GyrBAKKK (D211K/E212K/E213K/E214K), pH 7.5, 30°C Mycobacterium tuberculosis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
5.6.2.2 ATP + H2O Mycobacterium tuberculosis
-
 ADP + phosphate
-
 ?
5.6.2.2 ATP + H2O Mycobacterium tuberculosis H37Rv
-
 ADP + phosphate
-
 ?
5.6.2.2 ATP + H2O Mycobacterium tuberculosis ATCC 25618
-
 ADP + phosphate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
5.6.2.2 Mycobacterium tuberculosis P9WG47 AND P9WG45 subunits a and b
-
5.6.2.2 Mycobacterium tuberculosis ATCC 25618 P9WG47 AND P9WG45 subunits a and b
-
5.6.2.2 Mycobacterium tuberculosis H37Rv P9WG47 AND P9WG45 subunits a and b
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5.6.2.2 ATP + H2O
-
 Mycobacterium tuberculosis ADP + phosphate
-
 ?
5.6.2.2 ATP + H2O
-
 Mycobacterium tuberculosis H37Rv ADP + phosphate
-
 ?
5.6.2.2 ATP + H2O
-
 Mycobacterium tuberculosis ATCC 25618 ADP + phosphate
-
 ?
5.6.2.2 additional information negative DNA supercoiling activity assays using relaxed pBR322 DNA as substrate, and ATPase assays Mycobacterium tuberculosis ?
-
-
5.6.2.2 additional information negative DNA supercoiling activity assays using relaxed pBR322 DNA as substrate, and ATPase assays Mycobacterium tuberculosis H37Rv ?
-
-
5.6.2.2 additional information negative DNA supercoiling activity assays using relaxed pBR322 DNA as substrate, and ATPase assays Mycobacterium tuberculosis ATCC 25618 ?
-
-

Subunits

EC Number Subunits Comment Organism
5.6.2.2 heterodimer subunits GyrB and GyrA57 Mycobacterium tuberculosis
5.6.2.2 More quaternary organization and interdomain connections of full-length (GyrB-GyrA57)2 gyrase, structure analysis, detailed overview Mycobacterium tuberculosis

Synonyms

EC Number Synonyms Comment Organism
5.6.2.2 ATPase
-
 Mycobacterium tuberculosis
5.6.2.2 DNA gyrase
-
 Mycobacterium tuberculosis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
5.6.2.2 30
-
 ATPase assay at Mycobacterium tuberculosis
5.6.2.2 37
-
 negative DNA supercoiling activity assay at Mycobacterium tuberculosis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
5.6.2.2 0.05
-
 ATP wild-type enzyme GyrBA, pH 7.5, 30°C Mycobacterium tuberculosis
5.6.2.2 0.06
-
 ATP enzyme dimeric mutant GyrBA5, pH 7.5, 30°C Mycobacterium tuberculosis
5.6.2.2 0.06
-
 ATP enzyme dimeric mutant GyrBA57DELTAC-loop, pH 7.5, 30°C Mycobacterium tuberculosis
5.6.2.2 0.08
-
 ATP enzyme mutant GyrBAmC-loop (E221K/D225K/E228K/R236E/H244A), pH 7.5, 30°C Mycobacterium tuberculosis
5.6.2.2 0.09
-
 ATP enzyme mutant GyrBAKKK (D211K/E212K/E213K/E214K), pH 7.5, 30°C Mycobacterium tuberculosis
5.6.2.2 0.11
-
 ATP enzyme mutant GyrBADELTAC-loop, pH 7.5, 30°C Mycobacterium tuberculosis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
5.6.2.2 7.5
-
 assay at Mycobacterium tuberculosis

General Information

EC Number General Information Comment Organism
5.6.2.2 additional information the C-loop (residues 214-245), a Corynebacteriales-specific GyrB insert, stabilizes the a specific open N-gate conformation of the enzyme, structure overview. The N-gate of GyrBA57 (Mtb) gyrase lies in an extremely open conformation. The C-loop plays a role in the ATPase activity of the enzyme. The open N-gate state persists even in the DNA-free gyrase-AMPPNP complex and an unexpected connection between the ATPase and cleavage core domains mediated by two Corynebacteriales-specific motifs, respectively, the C-loop and DEEE-loop. The C-loop participates in the stabilization of this open conformation, explaining why this gyrase has a lower ATPase activity. Structure-function analysis, ATPase domain structure Mycobacterium tuberculosis
5.6.2.2 physiological function type II DNA topoisomerases are ubiquitous enzymes that manage DNA reorganization, and are therefore essential for many cellular events that take place during cell life. Using ATP as a cofactor, they control DNA topology by adding or removing DNA supercoils and by tangling or knotting DNA. Topoisomerases achieve these topological rearrangements through a succession of dissociation/association events of three domain-dimerization interfaces, which function as open/closed gates, to allow the movement of one DNA duplex through another Mycobacterium tuberculosis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
5.6.2.2 0.071
-
 ATP wild-type enzyme GyrBA, pH 7.5, 30°C Mycobacterium tuberculosis
5.6.2.2 0.075
-
 ATP enzyme dimeric mutant GyrBA5, pH 7.5, 30°C, pH 7.5, 30°C7 Mycobacterium tuberculosis
5.6.2.2 0.08
-
 ATP enzyme dimeric mutant GyrBA57DELTAC-loop, pH 7.5, 30°C Mycobacterium tuberculosis
5.6.2.2 0.09
-
 ATP enzyme mutant GyrBAKKK (D211K/E212K/E213K/E214K), pH 7.5, 30°C Mycobacterium tuberculosis
5.6.2.2 0.114
-
 ATP enzyme mutant GyrBAmC-loop (E221K/D225K/E228K/R236E/H244A), pH 7.5, 30°C Mycobacterium tuberculosis
5.6.2.2 0.367
-
 ATP enzyme mutant GyrBADELTAC-loop, pH 7.5, 30°C Mycobacterium tuberculosis