Pyrroloquinoline Quinone Ethanol Dehydrogenase in Methylobacterium extorquens AM1 Extends Lanthanide-Dependent Metabolism to Multicarbon Substrates

Abstract

Lanthanides are utilized by microbial methanol dehydrogenases, and it has been proposed that lanthanides may be important for other type I alcohol dehydrogenases. A triple mutant strain (mxaF xoxF1 xoxF2; named MDH-3), deficient in the three known methanol dehydrogenases of the model methylotroph Methylobacterium extorquens AM1, is able to grow poorly with methanol if exogenous lanthanides are added to the growth medium. When the gene encoding a putative quinoprotein ethanol dehydrogenase, exaF, was mutated in the MDH-3 background, the quadruple mutant strain could no longer grow on methanol in minimal medium with added lanthanum (La 3؉ IMPORTANCEExaF is the most efficient PQQ-dependent ethanol dehydrogenase reported to date and, to our knowledge, the first non-XoxFtype alcohol oxidation system reported to use lanthanides as a cofactor, expanding the importance of lanthanides in biochemistry and bacterial metabolism beyond methanol dehydrogenases to multicarbon metabolism. These results support an earlier proposal that an aspartate residue near the catalytic aspartate residue may be an indicator of rare-earth element utilization by type I alcohol dehydrogenases. Methylotrophy is the capability of organisms to metabolize reduced carbon compounds lacking carbon-carbon bonds as the sole source of carbon and energy (1). The genus Methylobacterium is comprised of aerobic facultative methylotrophs that can metabolize single-carbon compounds, such as methanol and methylamine, as well as multicarbon substrates like ethanol, acetate, ethylamine, pyruvate, and succinate (2, 3). Members of the genus Methylobacterium are wide-spread plant epiphytes (4, 5) that utilize their metabolic flexibility to gain an advantage in the phyllosphere, an oligotrophic environment with transient substrate availability (6, 7).Methanol dehydrogenase (MDH) is an essential enzyme for the methylotrophic metabolism of methanol and methane (8). In Gram-negative methylotrophic bacteria, MDHs are soluble, periplasmic proteins with pyrroloquinoline quinone (PQQ) as the prosthetic group (9, 10). The best studied PQQ-containing MDHs are ␣ 2 ␤ 2 tetramers consisting of the MxaF and MxaI proteins (11-14) that contain calcium (Ca 2ϩ ) in the active site (15, 16). Studies have provided evidence for the physiological role of a second type of PQQ-dependent MDH, XoxF, which has ϳ50% amino acid identity to MxaF from MxaFI-type MDHs (17). Metagenomic and environmental proteomics studies have demonstrated that xoxF is more widespread than mxaF in environmental samples (18)(19)(20)(21). Phylogenetic analysis of putative PQQ-containing MDHs has shown that XoxF-type MDHs are genetically diverse with at least five distinct clades, and it has been suggested that MxaFI-type MDHs represent a minor fraction of these MDHs (8,22). It has been further proposed that MxaFI-type MDHs may be the result of a second evolutionary event, with an ancestral XoxF-type MDH prototype (22). Together, these suppositions suggest that XoxFtype MDHs may be the primary MDHs for meth...

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

“…XoxF was purified from its native levels as described, by ammonium sulfate precipitation, cation‐exchange chromatography, and size‐exclusion chromatography (SEC), from M. extorquens AM1 cultures grown in the presence of 1 μ m LaCl 3 , CeCl 3 , or NdCl 3 (Figure S1 in the Supporting Information). The UV/visible spectra of these proteins are shown in Figure S2, with the characteristic absorption band at ≈360 nm and a shoulder at ≈410 nm indicative of the metal‐bound PQQ cofactor . The protein yields, metal contents, and A 280 nm / A 359 nm ratios for each XoxF were similar (Table S1), thus indicating similar cofactor loading.…”

“…This observation suggests that the coupling reagents in the dye‐linked assay, which possess lower reduction potentials than MxaG (Scheme A), cannot provide sufficient driving force for the Ca‐MDH reaction at neutral pH. By contrast, both dye‐linked and XoxG‐linked assays of Ln‐MDHs can be carried out at pH 7 . Together, these results suggest that either the reduction potential of the Ln III ‐PQQ cofactor in XoxFs is lower than that of the Ca II ‐PQQ cofactor in MxaFI, or the Ln‐MDH reaction requires less driving force to be provided by the electron acceptor.…”

Isolation and genomic characterization of Novimethylophilus kurashikiensis gen. nov. sp. nov., a new lanthanide‐dependent methylotrophic species of Methylophilaceae

“…Thus, XoxF is now recognized as the first Ln 3+ ‐dependent enzyme involved in bacterial methylotrophy. The ability of ExaF to use Ln 3+ as a cofactor, PQQ‐dependent ethanol dehydrogenase in Methylobacterium extorquens AM1, enlarged the importance of Ln 3+ to multicarbon metabolism (Good et al ., ). The finding of PedH, Ln 3+ ‐dependent PQQ‐alcohol dehydrogenase in a nonmethylotrophic bacterium, Pseudomonas putida KT2440, expanded the range of Ln 3+ utilizing bacteria beyond the methylotrophs (Wehrmann et al ., ).…”

Essenziell und weitverbreitet: Lanthanoid‐Metalloproteine

“…[26] Abbildung 3z eigt, dass besonders die früheren, häufiger vorkommenden Lanthanoide das Wachstum von Bakterien, die Ln-abhängige Enzyme exprimieren, unterstützen. [24,30] [34] Bemerkenswerterweise kann Formaldehyd in M. fumariolicum SolV direkt mittels MDH zu Ameisensäure und danach mithilfe von Formiat-Dehydrogenasen (FDHs) zu CO 2 umgewandelt werden. [27] Die Aufnahme und Biochemie der Lanthanoide sind vermutlich auf die häufiger vorkommenden, frühen Vertreter der Reihe zugeschnitten.…”

“…P. putida (PedH) isoliert wurden. [24,30] [34] Bemerkenswerterweise kann Formaldehyd in M. fumariolicum SolV direkt mittels MDH zu Ameisensäure und danach mithilfe von Formiat-Dehydrogenasen (FDHs) zu CO 2 umgewandelt werden. [14b] Der von Anthony und Zatman entwickelte Assay wird intensiv genutzt, um die Aktivitätv on Lanthanoid-abhängigen Enzymen zu bestimmen.…”