Ethylmalonyl Coenzyme A Mutase Operates as a Metabolic Control Point in Methylobacterium extorquens AM1

Good, Nathan; Martinez‐Gomez, N. Cecilia; Beck, David A. C.; Lidstrom, Mary E.

doi:10.1128/jb.02478-14

Cited by 12 publications

(15 citation statements)

References 36 publications

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“…coli TOP10 cells (Sigma-Aldrich, St. Louis, MO) were used for cloning and plasmid propagation as reported previously (42). All plasmids were verified by colony PCR and sequencing.…”

Section: Methodsmentioning

confidence: 99%

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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“…coli TOP10 cells (Sigma-Aldrich, St. Louis, MO) were used for cloning and plasmid propagation as reported previously (42). All plasmids were verified by colony PCR and sequencing.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2016

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“…Cells in the filter were frozen immediately with liquid nitrogen in a precooled 50-ml plastic tube. Samples were lyophilized for 2 h. Hot extraction of the metabolites was achieved as described by Good et al (33). GC/MS measurements were used to analyze amino acid and organic acid contents as previously described (23).…”

Section: Figmentioning

confidence: 99%

Methenyl-Dephosphotetrahydromethanopterin Is a Regulatory Signal for Acclimation to Changes in Substrate Availability in Methylobacterium extorquens AM1

2015

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During an environmental perturbation, the survival of a cell and its response to the perturbation depend on both the robustness and functionality of the metabolic network. The regulatory mechanisms that allow the facultative methylotrophic bacterium Methylobacterium extorquens AM1 to effect the metabolic transition from succinate to methanol growth are not well understood. Methenyl-dephosphotetrahydromethanopterin (methenyl-dH 4 MPT), an early intermediate during methanol metabolism, transiently accumulated 7-to 11-fold after addition of methanol to a succinate-limited culture. This accumulation partially inhibited the activity of the methylene-H 4 MPT dehydrogenase, MtdA, restricting carbon flux to the assimilation cycles. A strain overexpressing the gene (mch) encoding the enzyme that consumes methenyl-dH 4 MPT did not accumulate methenyl-dH 4 MPT and had a growth rate that was 2.7-fold lower than that of the wild type. This growth defect demonstrates the physiological relevance of this enzymatic regulatory mechanism during the acclimation period. Changes in metabolites and enzymatic activities were analyzed in the strain overexpressing mch. Under these conditions, the activity of the enzyme coupling formaldehyde with dH 4 MPT (Fae) remained constant, with concomitant formaldehyde accumulation. Release of methenyl-dH 4 MPT regulation did not affect the induction of the serine cycle enzyme activities immediately after methanol addition, but after 1 h, the activity of these enzymes decreased, likely due to the toxicity of formaldehyde accumulation. Our results support the hypothesis that in a changing environment, the transient accumulation of methenyl-dH 4 MPT and inhibition of MtdA activity are strategies that permit flexibility and acclimation of the metabolic network while preventing the accumulation of the toxic compound formaldehyde. IMPORTANCEThe identification and characterization of regulatory mechanisms for methylotrophy are in the early stages. We report a nontranscriptional regulatory mechanism that was found to operate as an immediate response for acclimation during changes in substrate availability. Methenyl-dH 4 MPT, an early intermediate during methanol oxidation, reversibly inhibits the methylene-H 4 MPT dehydrogenase, MtdA, when Methylobacterium extorquens is challenged to switch from succinate to methanol growth. Bypassing this regulatory mechanism causes formaldehyde to accumulate. Fae, the enzyme catalyzing the conversion of formaldehyde to methylene-dH 4 MPT, was also identified as another potential regulatory target using this strategy. The results herein further our understanding of the complex regulatory network in methylotrophy and will allow us to improve metabolic engineering strategies of methylotrophs for the production of value-added products. Methylobacterium extorquens AM1 is a facultative methylotroph that is capable of growth on both one-carbon (C 1 ) compounds, such as methanol and methylamine, and multicarbon compounds, such as succinate. Methanol metabolism is predic...

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“…50 mL cultures were grown in shake flasks with or without LaCl 3 to an OD 600 of 0.7 that correlated with mid-exponential growth. Total RNA samples were procured, and quality was verified as previously described (48). Two biological replicates were prepared for each condition.…”

Section: Methodsmentioning

confidence: 99%

Investigation of lanthanide-dependent methylotrophy uncovers complementary roles for alcohol dehydrogenase enzymes

Good

Moore

et al. 2018

Preprint

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15The discovery that methylotrophic bacteria can utilize lanthanides as catalysts for methanol 16 metabolism has opened new areas of biology and biochemistry. Recent studies of lanthanide-17 dependent enzymes have focused on biochemical and kinetic properties or the regulation of 18 encoding genes. Kinetic analysis of a pyrroloquinoline quinone methanol dehydrogenase, XoxF1 19 (MexAM1_1746), from the model methylotroph Methylobacterium extorquens AM1 confirms 20 the use of different lanthanides as cofactors and formaldehyde as a product of methanol 21 oxidation, showing that not all XoxF MDH produce formate as the only end product in vivo. The 22 30 pyrroloquinoline quinone alcohol dehydrogenase ExaF (MexAM1_1139), but not XoxF1, can 31 reduce formaldehyde toxicity when lanthanides are present, providing evidence of a role for 32ExaF during lanthanide-dependent methylotrophy. We conclude from these results that 33 lanthanide-dependent methylotrophy is more efficient than calcium-dependent methylotrophy 34 in M. extorquens AM1, and that this change is due, at least in part, to the lanthanide-dependent 35 enzymes XoxF1 and ExaF. 36 37 IMPORTANCE 38 Lanthanides serve as cofactors for pyrroloquinoline quinone containing alcohol dehydrogenase 39 enzymes in methylotrophic bacteria. The present study addresses a fundamental gap in our 40 understanding of how lanthanides impact metabolism, including a detailed assessment of the 41 metabolic modifications to accommodate enhanced efficiency during methylotrophy. Kinetic 42 characterization of XoxF1 provides a detailed description of the impact of diverse lanthanides 43 on catalytic function for a lanthanide-dependent methanol dehydrogenase. We further show 44 that the lanthanide-dependent ethanol dehydrogenase ExaF can oxidize formaldehyde in vivo, 45 revealing complementary roles for these enzymes. This study provides novel insight into the 46 effects of lanthanides on bacterial metabolism, highlighting the implementation of multiple, 47 redundant and complementary oxidation systems. 48 49 53 5). Thus far, Ln-ADH can be grouped by their phylogeny and primary substrate as either XoxF-54 type methanol dehydrogenases (MDH) or ExaF-type ethanol dehydrogenases (EtDH). Reports 55 have provided evidence for XoxF having roles as both a periplasmic methanol and 56 formaldehyde-oxidation system (6) and as a regulator of expression of the calcium (Ca) 57 dependent MxaFI MDH (6, 7). MxaFI MDH has been considered the canonical primary catalyst 58 for methanol oxidation in Gram-negative methylotrophs (8, 9). MxaFI is a heterotetramer that 59 contains PQQ as a prosthetic group and coordinates a calcium ion (10-12). The discovery that 60 Ln are incorporated into the active site of XoxF MDH in place of Ca, allowing catalytic function, 61 has prompted the reexamination of methanol oxidation in methylotrophic bacteria (3, 13, 14). 62 In addition, we reported the first Ln-dependent EtDH ExaF, showing that Ln can impact multi-63 carbon as well as one-carbon metabolism (5). T...

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Ethylmalonyl Coenzyme A Mutase Operates as a Metabolic Control Point in Methylobacterium extorquens AM1

Cited by 12 publications

References 36 publications

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

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

Methenyl-Dephosphotetrahydromethanopterin Is a Regulatory Signal for Acclimation to Changes in Substrate Availability in Methylobacterium extorquens AM1

Investigation of lanthanide-dependent methylotrophy uncovers complementary roles for alcohol dehydrogenase enzymes

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