PMID:31017712

Biochemical and Structural Characterization of XoxG and XoxJ and Their Roles in Lanthanide-Dependent Methanol Dehydrogenase Activity

XoxJ is a periplasmic binding protein encoded in the xox operon with xoxF and xoxG

"these systems comprise two other proteins, XoxG (a c -type cytochrome) and XoxJ (a periplasmic binding protein of unknown function), about which little is known"
Crystal structure of XoxJ reveals a large hydrophobic cleft suggesting role in XoxF activation

"the x-ray crystal structure of XoxJ reveals a large hydrophobic cleft and suggests a role in activation of XoxF"
XoxJ plays an analogous role to MxaJ in activating methanol dehydrogenase

"By extension, we presume that XoxJ plays an analogous role in Ln-MDH activation...Deletion of mxaJ disrupts activation of the Ca-MDH"
XoxJ is a member of the periplasmic binding protein family not obviously associated with transporters

"MxaJ was recently characterized as a member of the periplasmic binding protein (PBP) family – proteins typically associated with membrane-bound transport systems for small molecules – but its substrate is unknown"
The mechanism of MDH activation by XoxJ remains poorly understood

"An understanding of this mechanism would facilitate studies of Ln substitution in XoxF...the mechanism of activation of MDHs with PQQ and metal ions"

file:METEA/xoxJ/xoxJ-uniprot.txt

UniProt entry for xoxJ periplasmic binding protein

XoxJ contains an N-terminal signal peptide for periplasmic localization

"SIGNAL 1..26"
XoxJ contains a solute-binding protein family 3 domain

"Solute-binding protein family 3/N-terminal domain-containing protein"
XoxJ structure contains a conserved disulfide bond

"DISULFID 41..94"
XoxJ crystal structure solved at 2.27 Angstroms resolution (PDB 6ONP)

"X-RAY CRYSTALLOGRAPHY (2.27 ANGSTROMS)"

PMID:32728125

Gene products and processes contributing to lanthanide homeostasis and methanol metabolism in Methylorubrum extorquens AM1

Loss of xoxJ phenocopies loss of both xoxF1 and xoxF2, establishing genetic necessity for XoxF-dependent methanol oxidation under lanthanum

"loss of either xoxG or xoxJ was equivalent to loss of both xoxF1 and xoxF2"
ΔxoxJ has a quantitative growth defect even without lanthanum, and this is not due to impaired mxa promoter expression, arguing for a direct role

"the growth phenotypes observed for the xoxG and xoxJ mutants grown in the absence of La3+ were not due to impaired mxa expression"

PMID:30653750

Use of rare-earth elements in the phyllosphere colonizer Methylobacterium extorquens PA1

In the related strain PA1, a ΔxoxGJ strain has a strong La-dependent growth defect, reinforcing that the Xox accessory module is critical to REE-dependent methanol metabolism

"the ΔxoxGJ strain maintained responsiveness to La3+, resulting in a strong growth defect in the presence of La3+"

file:METEA/xoxJ/xoxJ-deep-research-falcon.md

Falcon deep research report on xoxJ (Methylorubrum extorquens AM1)

XoxJ is best supported as a periplasmic accessory/activation factor (PBP fold), not a catalytic enzyme

"XoxJ is best supported as a **periplasmic accessory/activation factor** (a periplasmic binding protein-like fold), not a catalytic enzyme"
The crystal structure reveals a large hydrophobic cavity/cleft (central cavity ~1750 cubic Angstroms) consistent with binding a large hydrophobic partner rather than a small metabolite

"reveals a **large hydrophobic cavity/cleft** (reported central cavity ~**1750 Å3**)"
Leading mechanistic model is chaperone-like activation in which XoxJ binds a hydrophobic region of partially folded apo-XoxF to facilitate cofactor insertion/maturation

"XoxJ may bind a hydrophobic region of **partially folded apo-XoxF** to facilitate **cofactor insertion and/or maturation**"
A specific PQQ-chaperone role was considered but NOT supported by biochemical tests, so ligand specificity remains a structure-informed hypothesis

"considered a **PQQ-chaperone** role but report that their biochemical tests did **not support** that specific hypothesis"
XoxJ was expressed in E. coli, purified from the periplasm, and crystallized at 2.27 Angstroms, confirming periplasmic localization

"XoxJ was expressed in *E. coli*, purified from the periplasm, and crystallized at 2.27 Å"
PDB entry for XoxJ is 6ONP (XoxG is 6ONQ)

"PDB entries reported for this system include **XoxJ: 6ONP**"
xoxJ is annotated as a periplasmic binding protein genetically coupled to lanthanide-dependent methanol oxidation via its colocalization with xoxF1 and xoxG in the xoxF1GJ operon

"genetically coupled to lanthanide-dependent methanol oxidation via its colocalization with xoxF1 and xoxG (xoxF1GJ)"