Literature summary for 1.1.2.10 extracted from

Featherston, E.R.; Mattocks, J.A.; Tirsch, J.L.; Cotruvo, J.A.
Heterologous expression, purification, and characterization of proteins in the lanthanome (2021), Methods Enzymol., 650, 119-157 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene xoxF, recombinant enzyme expression in Methylorubrum extorquens strain AM1 and in Escherichia coli. gene xoxJ, recombinant expression in Escherichia coli strain BL21(DE3)	Methylorubrum extorquens

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	when La-, Ce-, and Nd-metalated XoxFs are assayed with XoxG as electron acceptor, the Vmax values are not significantly different for the three enzymes, but the Km for XoxG increases from 0.0025 mM for La-XoxF to 0.0076 mM for Nd-XoxF	Methylorubrum extorquens
0.0025	-	cytochrome cGJ	pH 7.0, 22°C, La-XoxF	Methylorubrum extorquens
0.0076	-	cytochrome cGJ	pH 7.0, 22°C, Nd-XoxF	Methylorubrum extorquens

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
periplasm	-	Methylorubrum extorquens	-	-

Metals/Ions

Metals/Ions	Comment	Organism
Fe2+	in heme and bound to XoxG	Methylorubrum extorquens
La3+	-	Methylorubrum extorquens
additional information	lanthanides, especially the lighter and most abundant members (La, Ce, Pr, Nd, Sm, and Eu) of the lanthanide (Ln) series, are essential for catalysis in the most broadly distributed class of pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenases (MDHs). The number of distinct lanthanides supporting catalysis in vitro and/or in vivo differs from enzyme to enzyme: e.g. La-Nd, La-Sm/Eu, or La-Gd, according to the XoxF clade, in which an enzyme is found. Strain AM1 XoxF1 can be activated in vivo with La, Ce, Pr, and Nd, and poorly or not at all with Sm. The lanthanides are incorporated when they are added individually to the growth media, XoxF expressed in the presence of La, either from endogenous levels or recombinantly in a methylotroph, show roughly stoichiometric La incorporation, Nd incorporation is more variable. By contrast, plasmid-based expression of XoxF in the presence of Nd leads to substoichiometric Nd insertion into XoxF	Methylorubrum extorquens
Nd3+	-	Methylorubrum extorquens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
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	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 enzyme from Methylorubrum extorquens strain AM1 and from Escherichia coli, periplasmic extraction is easier in Escherichia coli than in Methylorubrum extorquens, by ammonium sulfate fractionation, cation exchange and hydrophobic interaction chromatography, and desalting gel filtration. Recombinant XoxJ from Escherichia coli strain BL21(DE3) periplasm by anion exchange chromatography, gel filtration, hydrophobic interaction chromatography, and ultrafiltration, followed by gel filtration, and cleavage of the signal peptide	Methylorubrum extorquens

Purification (Comment)

Organism

recombinant enzyme from Methylorubrum extorquens strain AM1 and from Escherichia coli, periplasmic extraction is easier in Escherichia coli than in Methylorubrum extorquens, by ammonium sulfate fractionation, cation exchange and hydrophobic interaction chromatography, and desalting gel filtration. Recombinant XoxJ from Escherichia coli strain BL21(DE3) periplasm by anion exchange chromatography, gel filtration, hydrophobic interaction chromatography, and ultrafiltration, followed by gel filtration, and cleavage of the signal peptide

Methylorubrum extorquens

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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
additional information	for assay of chromatography fractions during purifications, activity can be determined by reduction of 2,6-dichlorophenolindolphenol (DCPIP) using phenazine ethosulfate (PES) as an electron acceptor. Development of ddifferent assay methods, detailed overview	Methylorubrum extorquens	?	-	-
additional information	for assay of chromatography fractions during purifications, activity can be determined by reduction of 2,6-dichlorophenolindolphenol (DCPIP) using phenazine ethosulfate (PES) as an electron acceptor. Development of ddifferent assay methods, detailed overview	Methylorubrum extorquens NCIMB 9133	?	-	-
additional information	for assay of chromatography fractions during purifications, activity can be determined by reduction of 2,6-dichlorophenolindolphenol (DCPIP) using phenazine ethosulfate (PES) as an electron acceptor. Development of ddifferent assay methods, detailed overview	Methylorubrum extorquens DSM 1338	?	-	-
additional information	for assay of chromatography fractions during purifications, activity can be determined by reduction of 2,6-dichlorophenolindolphenol (DCPIP) using phenazine ethosulfate (PES) as an electron acceptor. Development of ddifferent assay methods, detailed overview	Methylorubrum extorquens ATCC 14718	?	-	-
additional information	for assay of chromatography fractions during purifications, activity can be determined by reduction of 2,6-dichlorophenolindolphenol (DCPIP) using phenazine ethosulfate (PES) as an electron acceptor. Development of ddifferent assay methods, detailed overview	Methylorubrum extorquens JCM 2805	?	-	-

Subunits

Subunits	Comment	Organism
?	x * 28600, recombinant XoxJ with cleaved signal peptide, SDS-PAGE	Methylorubrum extorquens
More	XoxF is encoded in an operon alongside genes encoding a c-type cytochrome, XoxG, the physiological electron acceptor for XoxF, as well as a periplasmic solute binding protein (SBP) XoxJ	Methylorubrum extorquens

Synonyms

Synonyms	Comment	Organism
methanol dehydrogenase	-	Methylorubrum extorquens
XoxF	-	Methylorubrum extorquens
XoxJ	-	Methylorubrum extorquens

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
22	-	assay at room temperature	Methylorubrum extorquens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	assay at	Methylorubrum extorquens
9	-	dye-linked assay at	Methylorubrum extorquens

Cofactor

Cofactor	Comment	Organism
cytochrome cGJ	c-type cytochrome, XoxG, contains a heme-binding pocket. Recombinant expression in Escherichia coli strain BL21(DE3) and purification from periplasm, method overview. When La-, Ce-, and Nd-metalated XoxFs are assayed with XoxG as electron acceptor, the Vmax values are not significantly different for the three enzymes, but the Km for XoxG increases from 0.0025 mM for La-XoxF to 0.0076 mM for Nd-XoxF. Determination of extinction coefficients and redox potential	Methylorubrum extorquens
heme	bound to XoxG	Methylorubrum extorquens
pyrroloquinoline quinone	PQQ, recombinant expression and binding analysis, method overview	Methylorubrum extorquens

General Information

General Information	Comment	Organism
evolution	XoxJ, encoded by the core Ln-MDH operon, is a member of the periplasmic (or solute) binding protein (PBP or SBP) family	Methylorubrum extorquens
additional information	XoxF is encoded in an operon alongside genes encoding a c-type cytochrome, XoxG, the physiological electron acceptor for XoxF, as well as a periplasmic solute binding protein (SBP) XoxJ. The crystal structure of XoxJ reveals general architectures similar to classic SBPs, except it exhibits an exceptionally large cavity, putatively for substrate binding, as well as a beta-sheet missing several strands	Methylorubrum extorquens