Structure and function of the lanthanide-dependent methanol dehydrogenase XoxF from the methanotroph Methylomicrobium buryatense 5GB1C

Abstract

In methylotrophic bacteria, which use one-carbon (C1) compounds as a carbon source, methanol is oxidized by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH) enzymes. Methylotrophic genomes generally encode two distinct MDHs, MxaF and XoxF. MxaF is a well-studied, calcium-dependent heterotetrameric enzyme whereas XoxF is a lanthanide-dependent homodimer. Recent studies suggest that XoxFs are likely the functional MDHs in many environments. In methanotrophs, methylotrophs that utilize methane, interactions between particulate methane monooxygenase (pMMO) and MxaF have been detected. To investigate the possibility of interactions between pMMO and XoxF, XoxF was isolated from the methanotroph Methylomicrobium buryatense 5GB1C (5G-XoxF). Purified 5G-XoxF exhibits a specific activity of 0.16 μmol DCPIP reduced min mg. The 1.85 Å resolution crystal structure reveals a La(III) ion in the active site, in contrast to the calcium ion in MxaF. The overall fold is similar to other MDH structures, but 5G-XoxF is a monomer in solution. An interaction between 5G-XoxF and its cognate pMMO was detected by biolayer interferometry, with a K value of 50 ± 17 μM. These results suggest an alternative model of MDH-pMMO association, in which a XoxF monomer may bind to pMMO, and underscore the potential importance of lanthanide-dependent MDHs in biological methane oxidation.

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

“…The large number of hydrophobic residues in the putative ligand‐binding region of XoxJ is a shared feature of the active sites of MDHs. In MDHs, the PQQ cofactor is stacked between a conserved tryptophan residue and an unusual disulfide bond that forms between adjacent cysteine residues (Figure S9) . Given the presumed necessity of a PQQ chaperone, we considered PQQ as a potential ligand for XoxJ, which also contains a tryptophan side chain in the central cavity (Trp200, Figure B).…”

“…These observations indicate that removing the cofactor disrupts the dimer interface, and implies that the reverse reaction, cofactor insertion, should be accompanied by an ordering of this region of the protein and dimerization. Based on conserved structures of MDHs in general, and a homology model of M. extorquens XoxF derived from the X‐ray structure of M. fumariolicum XoxF, more specifically, we speculate that the C terminus of the protein may undergo the most significant conformation change upon holoenzyme assembly. This region forms a helical domain that clamps over the active site, near the PQQ cofactor, in the homology model template structure (Figure S13).…”

“…Recently, it was discovered that many methylotrophs also encode MDHs and other PQQ‐dependent alcohol dehydrogenases that use a lanthanide ion (Ln III ) rather than calcium in their active sites. The Ln‐dependent MDH, XoxF, contains an analogous active site, except with the PQQ cofactor coordinated to a Ln III ion and an additional carboxylate ligand to the metal ion . Presumably, the greater Lewis acidity of Ln III ions over that of Ca II is advantageous for increasing the reduction potential of PQQ to facilitate this reaction …”

Electrocatalysis of a Europium‐Dependent Bacterial Methanol Dehydrogenase with Its Physiological Electron‐Acceptor Cytochrome cGJ

“…[7a] This extra ligandi sa ble to satisfy the higherc oordination number preferenceo ft he Ln 3 + ion compared to Ca 2 + .I th as been reported that XoxF-MDHs show highera ffinity and catalytic activity towardsaw ide range of primary alcohols compared to the MxaFI-MDHs due to the stronger Lewis acid characteristics of the Ln 3 + ion. [12] The field of lanthanide-dependent bacterial metabolism is quicklyg rowing. [7a, 10] Among XoxF-MDHs, in the present study we were particularly interested in the Eu 3 + dependent MDH from Methylacidiphilum fumariolicum SolV.V ery recentlyt he crystal structures of this MDH wered etermined in the presence of Eu 3 + and Ce 3 + .…”

Essenziell und weitverbreitet: Lanthanoid‐Metalloproteine

“…In diesem Fall war die Elektronendichte im PQQ-basierten Molekülorbital über das aromatische System Abbildung 4. Vergleich der aktiven Zentren von Ca-MDH [32] (M. extorquens,P DB:1 W6S) und La-MDH [31] (Mm. buryatense,P DB:6 DAM).…”