XoxF-Type Methanol Dehydrogenase from the Anaerobic Methanotroph “Candidatus Methylomirabilis oxyfera”

Wu, Ming; Wessels, Hans; Pol, Arjan; Camp, Huub J. M. Op den; Jetten, Mike S. M.; Niftrik, Laura van; Keltjens, Jan T.

doi:10.1128/aem.03292-14

Appl Environ Microbiol

2015

DOI: 10.1128/aem.03292-14

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XoxF-Type Methanol Dehydrogenase from the Anaerobic Methanotroph “Candidatus Methylomirabilis oxyfera”

Ming Wu

Hans Wessels

Arjan Pol

et al.

Abstract: b "Candidatus Methylomirabilis oxyfera" is a newly discovered anaerobic methanotroph that, surprisingly, oxidizes methane through an aerobic methane oxidation pathway. The second step in this aerobic pathway is the oxidation of methanol. In Gramnegative bacteria, the reaction is catalyzed by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH). The genome of "Ca. Methylomirabilis oxyfera" putatively encodes three different MDHs that are localized in one large gene cluster: one so-called MxaFI-… Show more

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Cited by 67 publications

(60 citation statements)

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“…In 2012, this was resolved when Nakagawa et al demonstrated that MeDH, purified from M. extorquens AM1 cells grown in medium containing La, consisted of a XoxF dimer containing 1.24 atoms of La and lacked Ca (11). Since these findings, XoxF has been purified from a variety of methylotrophs, displaying differences in subunit composition, optimal enzyme assay parameters, and oxidation products depending on the organism studied (9,10,25). In 2014, Pol et al addressed the biochemical promiscuity of XoxF MeDHs by determining that a variety of lanthanides, including La, Ce, Pr, and Nd, could support methanol-dependent growth of Methylacidiphilum fumariolicum SolV (8).…”

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confidence: 99%

“…A key step in this process is the oxidation of methanol to formaldehyde, which is carried out by different enzymes, including methanol dehydrogenase (MeDH) and alcohol oxidase, depending on the specific methylotroph (6,7). Recently, it was discovered that some types of MeDHs require rareearth elements, specifically lanthanides, as cofactors (8)(9)(10)(11) [Yb], and lutetium [Lu]) and two chemically similar elements (scandium [Sc] and yttrium [Y]). The term "rare earth" is deceptive, as these lanthanide elements are relatively abundant in the Earth's crust, found at levels similar to those seen for copper and zinc (Ce, 66 ppm; La 39 ppm; Cu, 60 ppm, Zn, 70 ppm) (12).…”

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Lanthanide-Dependent Regulation of Methanol Oxidation Systems in Methylobacterium extorquens AM1 and Their Contribution to Methanol Growth

et al. 2016

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Methylobacterium extorquens AM1 has two distinct types of methanol dehydrogenase (MeDH) enzymes that catalyze the oxidation of methanol to formaldehyde. MxaFI-MeDH requires pyrroloquinoline quinone (PQQ) and Ca in its active site, while XoxFMeDH requires PQQ and lanthanides, such as Ce and La. Using MeDH mutant strains to conduct growth analysis and MeDH activity assays, we demonstrate that M. extorquens AM1 has at least one additional lanthanide-dependent methanol oxidation system contributing to methanol growth. Additionally, the abilities of different lanthanides to support growth were tested and strongly suggest that both XoxF and the unknown methanol oxidation system are able to use La, Ce, Pr, Nd, and, to some extent, Sm. Further, growth analysis using increasing La concentrations showed that maximum growth rate and yield were achieved at and above 1 M La, while concentrations as low as 2.5 nM allowed growth at a reduced rate. Contrary to published data, we show that addition of exogenous lanthanides results in differential expression from the xox1 and mxa promoters, upregulating genes in the xox1 operon and repressing genes in the mxa operon. Using transcriptional reporter fusions, intermediate expression from both the mxa and xox1 promoters was detected when 50 to 100 nM La was added to the growth medium, suggesting that a condition may exist under which M. extorquens AM1 is able to utilize both enzymes simultaneously. Together, these results suggest that M. extorquens AM1 actively senses and responds to lanthanide availability, preferentially utilizing the lanthanide-dependent MeDHs when possible. IMPORTANCEThe biological role of lanthanides is a nascent field of study with tremendous potential to impact many areas in biology. Our studies demonstrate that there is at least one additional lanthanide-dependent methanol oxidation system, distinct from the MxaFI and XoxF MeDHs, that may aid in classifying additional environmental organisms as methylotrophs. Further, our data suggest that M. extorquens AM1 has a mechanism to regulate which MeDH is transcribed, depending on the presence or absence of lanthanides. While the mechanism controlling differential regulation is not yet understood, further research into how methylotrophs obtain and use lanthanides will facilitate their cultivation in the laboratory and their use as a biomining and biorecycling strategy for recovery of these commercially valuable rare-earth elements. Methylotrophs have gained worldwide interest as platforms for the production of value-added chemicals from singlecarbon compounds, turning atmospheric pollutants like methane and methanol into green chemicals, including biofuels and biodegradable plastics (1-5). A key step in this process is the oxidation of methanol to formaldehyde, which is carried out by different enzymes, including methanol dehydrogenase (MeDH) and alcohol oxidase, depending on the specific methylotroph (6, 7). Recently, it was discovered that some types of MeDHs require rareearth elements, specifically lantha...

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Lanthanide-Dependent Regulation of Methanol Oxidation Systems in Methylobacterium extorquens AM1 and Their Contribution to Methanol Growth

et al. 2016

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“…It was initially believed that this enzyme was critical for methylotrophic growth on methanol since no methanol dehydrogenase activity was observed in mutants defective in the production of this protein (24). Subsequently, however, it was found that there is a homolog to the large subunit, termed XoxF, with 50% sequence identity to MxaF (25,26). This also encodes a PQQ-dependent methanol dehydrogenase that is associated with the periplasm (26, 27) but appears to be composed only of a single subunit with a predicted mass of 65 kDa (28) or associated with the small subunit of MxaI (26), depending on the microbe.…”

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confidence: 99%

“…This also encodes a PQQ-dependent methanol dehydrogenase that is associated with the periplasm (26, 27) but appears to be composed only of a single subunit with a predicted mass of 65 kDa (28) or associated with the small subunit of MxaI (26), depending on the microbe. Further, it is often observed that multiple homologs of XoxF are found in the genome of a variety of methylotrophs and methanotrophs (25,26,29,30).…”

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Cerium Regulates Expression of Alternative Methanol Dehydrogenases in Methylosinus trichosporium OB3b

Haque

Kalidass

Bandow

et al. 2015

Appl Environ Microbiol

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bMethanotrophs have multiple methane monooxygenases that are well known to be regulated by copper, i.e., a "copper switch." At low copper/biomass ratios the soluble methane monooxygenase (sMMO) is expressed while expression and activity of the particulate methane monooxygenase (pMMO) increases with increasing availability of copper. In many methanotrophs there are also multiple methanol dehydrogenases (MeDHs), one based on Mxa and another based on Xox. Mxa-MeDH is known to have calcium in its active site, while Xox-MeDHs have been shown to have rare earth elements in their active site. We show here that the expression levels of Mxa-MeDH and Xox-MeDH in Methylosinus trichosporium OB3b significantly decreased and increased, respectively, when grown in the presence of cerium but the absence of copper compared to the absence of both metals. Expression of sMMO and pMMO was not affected. In the presence of copper, the effect of cerium on gene expression was less significant, i.e., expression of Mxa-MeDH in the presence of copper and cerium was slightly lower than in the presence of copper alone, but Xox-MeDH was again found to increase significantly. As expected, the addition of copper caused sMMO and pMMO expression levels to significantly decrease and increase, respectively, but the simultaneous addition of cerium had no discernible effect on MMO expression. As a result, it appears Mxa-MeDH can be uncoupled from methane oxidation by sMMO in M. trichosporium OB3b but not from pMMO. It is well known that microorganisms have diverse mechanisms to sense and respond to metals in their environment. These mechanisms typically include strategies to regulate gene expression in response to the presence or absence of metals such as copper, zinc, iron, manganese, arsenic, and mercury (see, for example, references 1, 2, 3 and 4). One such phenomenon is the "copper switch" in methanotrophs. That is, these microbes utilize methane as their sole growth substrate but have two different monooxygenases for the initial oxidation of methane to methanol. One, the particulate methane monooxygenase (pMMO), is found in the intracytoplasmic membranes of these microbes, and its expression and activity increases with increasing availability of copper. The second, the soluble methane monooxygenase (sMMO), is found in the cytoplasm and is only expressed when copper is unavailable (5). These two forms of MMO have very different structures, activities, and substrate ranges (5-14), and so careful consideration of the form of MMO expressed is critical for understanding methanotrophic ecology, as well for various applications of methanotrophy, including the removal of chlorinated solvents and methane, a potent greenhouse gas (5, 10, 12, 15).Further, interest in the commercial application of methanotrophy has dramatically accelerated in recent years, in part due to increased methane supplies given advances in hydraulic fracturing of shale formations. As a result, methane prices have become quite low, with the wellhead price of natural gas dropping fro...

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“…Together, these suppositions suggest that XoxFtype MDHs may be the primary MDHs for methylotrophy. For the few that have been studied to date, production of functional XoxF-type MDHs requires the presence of lanthanides in the growth medium (23)(24)(25)(26)(27) and all reported XoxF-type MDHs that have been biochemically characterized thus far contain a lanthanide such as cerium (Ce 3ϩ ) or lanthanum (La 3ϩ ), rather than Ca 2ϩ in the active site (25,27). Limited kinetic studies of XoxFtype MDHs have shown an increased efficiency when oxidizing methanol compared to that of MxaFI-type MDHs (summarized in reference 22).…”

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

et al. 2016

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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...

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XoxF-Type Methanol Dehydrogenase from the Anaerobic Methanotroph “Candidatus Methylomirabilis oxyfera”

Cited by 67 publications

References 70 publications

Lanthanide-Dependent Regulation of Methanol Oxidation Systems in Methylobacterium extorquens AM1 and Their Contribution to Methanol Growth

Lanthanide-Dependent Regulation of Methanol Oxidation Systems in Methylobacterium extorquens AM1 and Their Contribution to Methanol Growth

Cerium Regulates Expression of Alternative Methanol Dehydrogenases in Methylosinus trichosporium OB3b

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

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