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Literature summary for 1.1.2.10 extracted from

  • Featherston, E.R.; Rose, H.R.; McBride, M.J.; Taylor, E.M.; Boal, A.K.; Cotruvo, J.A.
    Biochemical and structural characterization of XoxG and XoxJ and their roles in lanthanide-dependent methanol dehydrogenase activity (2019), ChemBioChem, 20, 2360-2372 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
gene xoxJ, recombinant expression in Escherichia coli Methylorubrum extorquens

Crystallization (Commentary)

Crystallization (Comment) Organism
purified wild-type XoxJ, X-ray diffraction structure determination and analysis at 2.27 A resolution, single-wavelength anomalous diffraction method, molecular replacement method and modeling, soaking of PQQ into XoxJ crystals is unsuccessful Methylorubrum extorquens

Protein Variants

Protein Variants Comment Organism
W200F site directed mutagenesis, the variant of XoxJ does not bind cofactor PQQ in contrast to the wild-type enzyme Methylorubrum extorquens

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0025
-
oxidized cytochrome cL in the presence of La3+, at pH 7.0 and 30°C Methylorubrum extorquens
0.0044
-
oxidized cytochrome cL in the presence of Ce3+, at pH 7.0 and 30°C Methylorubrum extorquens
0.0076
-
oxidized cytochrome cL in the presence of Nd3+, at pH 7.0 and 30°C Methylorubrum extorquens
0.018
-
methanol in the presence of Nd3+, at pH 7.0 and 30°C Methylorubrum extorquens
0.022
-
methanol in the presence of La3+, at pH 7.0 and 30°C Methylorubrum extorquens
0.049
-
methanol in the presence of Ce3+, at pH 7.0 and 30°C Methylorubrum extorquens

Metals/Ions

Metals/Ions Comment Organism Structure
Ce3+ dependent on Methylorubrum extorquens
Ce3+ a lanthanide is required Methylorubrum extorquens
La3+ dependent on Methylorubrum extorquens
La3+ a lanthanide is required Methylorubrum extorquens
additional information XoxF has maximal activity in the standard artificial dye-linked assay when metallated with Pr and Nd. Activity is about 30% lower with La and falls off quickly beyond Nd. This biphasic behavior is attributed to competition between the Lewis acidity of the LnIII ion, increasing across the series and therefore enhancing reactivity of the pyrroloquinoline quinone (PQQ) cofactor, with other, opposing factors Methylorubrum extorquens
Nd3+ dependent on Methylorubrum extorquens
Nd3+ a lanthanide is required Methylorubrum extorquens

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
methanol + 2 cytochrome cGJ Methylorubrum extorquens
-
formaldehyde + 2 reduced cytochrome cGJ
-
r
methanol + 2 cytochrome cGJ Methylorubrum extorquens NCIMB 9133
-
formaldehyde + 2 reduced cytochrome cGJ
-
r
methanol + 2 cytochrome cGJ Methylorubrum extorquens DSM 1338
-
formaldehyde + 2 reduced cytochrome cGJ
-
r
methanol + 2 cytochrome cGJ Methylorubrum extorquens ATCC 14718
-
formaldehyde + 2 reduced cytochrome cGJ
-
r
methanol + 2 cytochrome cGJ Methylorubrum extorquens JCM 2805
-
formaldehyde + 2 reduced cytochrome cGJ
-
r

Organism

Organism UniProt Comment Textmining
Methylorubrum extorquens
-
-
-
Methylorubrum extorquens P16027 AND P14775 AND P14774 subunit 1/MoxF/XoxF, subunit 2/moxI/XoxJ, and cytochrome cL/moxG/XoxG; Methylobacterium extorquens
-
Methylorubrum extorquens ATCC 14718 P16027 AND P14775 AND P14774 subunit 1/MoxF/XoxF, subunit 2/moxI/XoxJ, and cytochrome cL/moxG/XoxG; Methylobacterium extorquens
-
Methylorubrum extorquens DSM 1338 P16027 AND P14775 AND P14774 subunit 1/MoxF/XoxF, subunit 2/moxI/XoxJ, and cytochrome cL/moxG/XoxG; Methylobacterium extorquens
-
Methylorubrum extorquens JCM 2805 P16027 AND P14775 AND P14774 subunit 1/MoxF/XoxF, subunit 2/moxI/XoxJ, and cytochrome cL/moxG/XoxG; Methylobacterium extorquens
-
Methylorubrum extorquens NCIMB 9133 P16027 AND P14775 AND P14774 subunit 1/MoxF/XoxF, subunit 2/moxI/XoxJ, and cytochrome cL/moxG/XoxG; Methylobacterium extorquens
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type XoxJ from Escherichia coli by anion exchange and hydrophobic interaction chromatography, and gel filtration. Native subunit XoxF is purified by ammonium sulfate fractionation, cation exchange chromatography, and gel filtration Methylorubrum extorquens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
methanol + 2 cytochrome cGJ
-
Methylorubrum extorquens formaldehyde + 2 reduced cytochrome cGJ
-
r
methanol + 2 cytochrome cGJ
-
Methylorubrum extorquens NCIMB 9133 formaldehyde + 2 reduced cytochrome cGJ
-
r
methanol + 2 cytochrome cGJ
-
Methylorubrum extorquens DSM 1338 formaldehyde + 2 reduced cytochrome cGJ
-
r
methanol + 2 cytochrome cGJ
-
Methylorubrum extorquens ATCC 14718 formaldehyde + 2 reduced cytochrome cGJ
-
r
methanol + 2 cytochrome cGJ
-
Methylorubrum extorquens JCM 2805 formaldehyde + 2 reduced cytochrome cGJ
-
r
methanol + 2 oxidized cytochrome cL c-type cytochrome XoxG Methylorubrum extorquens formaldehyde + 2 reduced cytochrome cL
-
?
methanol + phenazine ethosulfate
-
Methylorubrum extorquens formaldehyde + reduced phenazine ethosulfate
-
?
additional information MDH activity is also spectrophotometrically monitored via the reduction of 2,6-dichlorophenolindophenol (DCPIP) by the MDH with either phenazine ethosulfate or phenazine methosulfate as mediator. XoxG exhibits an unusually low reduction potential. The reduction potential of XoxG may be specifically optimized for transfer of electrons from PQQ, bound to lighter LnIIIs, to the cytochrome Methylorubrum extorquens ?
-
-
additional information MDH activity is also spectrophotometrically monitored via the reduction of 2,6-dichlorophenolindophenol (DCPIP) by the MDH with either phenazine ethosulfate or phenazine methosulfate as mediator. XoxG exhibits an unusually low reduction potential. The reduction potential of XoxG may be specifically optimized for transfer of electrons from PQQ, bound to lighter LnIIIs, to the cytochrome Methylorubrum extorquens NCIMB 9133 ?
-
-
additional information MDH activity is also spectrophotometrically monitored via the reduction of 2,6-dichlorophenolindophenol (DCPIP) by the MDH with either phenazine ethosulfate or phenazine methosulfate as mediator. XoxG exhibits an unusually low reduction potential. The reduction potential of XoxG may be specifically optimized for transfer of electrons from PQQ, bound to lighter LnIIIs, to the cytochrome Methylorubrum extorquens DSM 1338 ?
-
-
additional information MDH activity is also spectrophotometrically monitored via the reduction of 2,6-dichlorophenolindophenol (DCPIP) by the MDH with either phenazine ethosulfate or phenazine methosulfate as mediator. XoxG exhibits an unusually low reduction potential. The reduction potential of XoxG may be specifically optimized for transfer of electrons from PQQ, bound to lighter LnIIIs, to the cytochrome Methylorubrum extorquens ATCC 14718 ?
-
-
additional information MDH activity is also spectrophotometrically monitored via the reduction of 2,6-dichlorophenolindophenol (DCPIP) by the MDH with either phenazine ethosulfate or phenazine methosulfate as mediator. XoxG exhibits an unusually low reduction potential. The reduction potential of XoxG may be specifically optimized for transfer of electrons from PQQ, bound to lighter LnIIIs, to the cytochrome Methylorubrum extorquens JCM 2805 ?
-
-

Synonyms

Synonyms Comment Organism
Ce-XoxF
-
Methylorubrum extorquens
La-XoxF
-
Methylorubrum extorquens
lanthanide-dependent MDH
-
Methylorubrum extorquens
lanthanide-dependent methanol dehydrogenase
-
Methylorubrum extorquens
Ln-dependent MDH
-
Methylorubrum extorquens
Ln-dependent methanol dehydrogenase
-
Methylorubrum extorquens
Nd-XoxF
-
Methylorubrum extorquens
XoxF
-
Methylorubrum extorquens
XoxJ
-
Methylorubrum extorquens

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Methylorubrum extorquens

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.2
-
oxidized cytochrome cL in the presence of Ce3+, at pH 7.0 and 30°C Methylorubrum extorquens
1.2
-
oxidized cytochrome cL in the presence of La3+, at pH 7.0 and 30°C Methylorubrum extorquens
1.2
-
methanol in the presence of Nd3+, at pH 7.0 and 30°C Methylorubrum extorquens
1.2
-
cytochrome cGJ Ce-XoxF, pH 7.0, 30°C, determined using the XoxG/cyt c-linked activity Methylorubrum extorquens
1.2
-
cytochrome cGJ La-XoxF, pH 7.0, 30°C, determined using the XoxG/cyt c-linked activity Methylorubrum extorquens
1.2
-
methanol Nd-XoxF, pH 7.0, 30°C, determined using the dye-linked activity, DCPIP reduction Methylorubrum extorquens
1.3
-
oxidized cytochrome cL in the presence of Nd3+, at pH 7.0 and 30°C Methylorubrum extorquens
1.3
-
cytochrome cGJ Nd-XoxF, pH 7.0, 30°C, determined using the XoxG/cyt c-linked activity Methylorubrum extorquens
2.8
-
methanol in the presence of Ce3+, at pH 7.0 and 30°C Methylorubrum extorquens
2.8
-
methanol Ce-XoxF, pH 7.0, 30°C, determined using the dye-linked activity, DCPIP reduction Methylorubrum extorquens
4
-
methanol in the presence of La3+, at pH 7.0 and 30°C Methylorubrum extorquens
4
-
methanol La-XoxF, pH 7.0, 30°C, determined using the dye-linked activity, DCPIP reduction Methylorubrum extorquens

Cofactor

Cofactor Comment Organism Structure
cytochrome cGJ XoxG exhibits an unusually low reduction potential with impact on physiological methanol oxidation, XoxG crystal structure and structure-function analysis. The heme c moiety, which is covalently attached to the protein through two thioether bonds to C95 and C98 via the signature CXXCH motif for heme attachment, is enclosed in a hydrophobic pocket formed by three core alpha-helices (helices I, III, and V). This binding motif leaves one of the heme edges open to solvent. Typical of most class I c-type cytochromes, the FeIII is axially ligated by a His residue contributed by helix I (H99) and a Met residue from the loop between helices III and V (M143). Unlike most other class I cytochromes c, XoxG lacks helix IV, and this region is instead a 19-residue loop, the end of which is partially disordered. Alignment of XoxG with the cytochrome c domain and the XoxF homology model with the dehydrogenase domain suggests a plausible XoxF binding interface on XoxG. In the model, the loops between helices I and II and between III and V in XoxG are positioned to interact with XoxF. The X-ray crystal structure of XoxG is solved to 2.71 A resolution Methylorubrum extorquens
pyrroloquinoline quinone PQQ, PQQ is synthesized in the cytosol, but the proteins that use it are periplasmic, it might bind to subunit XoxJ near or at residue W200, no binding to XoxJ mutant W200F. Metal-bound PQQ is bound at subunit XoxF Methylorubrum extorquens

General Information

General Information Comment Organism
evolution XoxJ are predicted to be members of the periplasmic binding protein (PBP) family Methylorubrum extorquens
physiological function lanthanide (Ln)-dependent methanol dehydrogenases (MDHs) have been recently shown to be widespread in methylotrophic bacteria. Along with the core MDH protein, XoxF, these systems comprise two other proteins, XoxG (a c-type cytochrome) and XoxJ (a periplasmic binding protein of unknown function) in methyltroph, Methylobacterium extorquens strain AM1. In contrast to results obtained via an artificial assay system, assays of XoxFs metallated with LaIII, CeIII, and NdIII using their physiological electron acceptor, XoxG, display Ln-independent activities, the Km for XoxG markedly increases from La to Nd. This result suggests that XoxG's redox properties are tuned specifically for lighter Lns in XoxF, an interpretation supported by the unusually low reduction potential of XoxG (+172 mV). The reduction potential of isolated XoxG measured may reasonably approximate the potential of the cytochrome in complex with XoxF Methylorubrum extorquens

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
57
-
methanol in the presence of Ce3+, at pH 7.0 and 30°C Methylorubrum extorquens
67
-
methanol in the presence of Nd3+, at pH 7.0 and 30°C Methylorubrum extorquens
170
-
oxidized cytochrome cL in the presence of Nd3+, at pH 7.0 and 30°C Methylorubrum extorquens
190
-
methanol in the presence of La3+, at pH 7.0 and 30°C Methylorubrum extorquens
280
-
oxidized cytochrome cL in the presence of Ce3+, at pH 7.0 and 30°C Methylorubrum extorquens
490
-
oxidized cytochrome cL in the presence of La3+, at pH 7.0 and 30°C Methylorubrum extorquens