Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
Biochemistry
Non Technical Summary
Problem Statement:Methane (CH4) is a concerning greenhouse gas with a ~25-fold higher global warming potential than carbon dioxide (CO2). Additionally, as the primary component of natural gas, methane accounts for nearly a quarter of energy consumed in the US. and has future potential as a renewable energy source through its controlled production from biomass.1 Therefore, understanding the details of methane production is important from both climate science and alternative energy perspectives. The major source of methane on Earth is from the biological process of methanogenesis, which produces over a billion tons of methane each year and accounts for about 70% of currently generated methane.2 The organisms carrying out methanogenesis are known as methanogens, which are anaerobic microbes comprising the largest group of organisms within the Archaea,3 the most ancient and least well understood domain of life. Although these organisms have been the source of intense research for many decades, many questions still remain regarding their unique energy metabolisms and other unusual biochemical pathways. Therefore, this research will functionally and mechanistically characterize a specific set of unique enzymes in methanogens to improve our understanding of the complex biochemistry occurring in these organisms, providing the fundamental knowledge necessary to take advantage of these enzymes in the future as targets for methane mitigation or for bioenergy applications.Relevance to advancing Virginia and the US:Methanogens are widespread in diverse anaerobic environments, including marine and freshwater habitats, anoxic soils, wastewater treatment facilities, and as important components of the microbiomes of animals, especially ruminants. In most cases, methanogens act in the final step of the degradation of organic matter, where bacteria consume more complex organic molecules and methanogens consume the end products of this bacterial catabolism (mainly H2 and CO2) to generate methane. In ruminants such as cattle, methanogens are a component of the rumen microbiome, which aids in the digestion of various feedstuffs. This is especially relevant in Virginia, since beef and dairy cattle combined are the second highest farm commodity in the Commonwealth, generating at least $400,000,000 in revenue each year.4 Unfortunately, methane is a potent greenhouse gas, and thus its release from ruminants via methanogenesis is a major concern. At least 50% of current global methane emissions are from livestock in the agricultural sector.5 Since climate conditions impact the quality and quantity of global food supplies, increasing methane concentrations is also a problem for long-term food availability and nutrition.6 Therefore, it is essential to develop strategies for reducing methane emissions, while not compromising livestock health, to sustain our health, environment, and economy in Virginia and beyond.Approach: To determine the function of unique genes/enzymes in methanogens, we will generate deletion strains of these genes in our model methanogens and analyze the metabolite profile of the deletion strain compared to the wild-type strain. This will allow us to understand the importance of the genes in a physiological context. Additionally, the enzymes will be studied in a purified form to reveal the chemical details of the reaction that they catalyze. Understanding these details is critical for potentially inhibiting these enzymes in the future to control methane production.Anticipated outcomes and impacts:This research will elucidate the functions and chemical mechanisms of unique enzymes in methanogens. The knowledge will provide the foundation for the future development of novel methane mitigation strategies, as well as for harnessing methanogenesis for generating liquid fuels and other useful chemicals. The research will engage several students and will be shared with the community to increase awareness about the significance of methane in our environment.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
The goals of this project are to determine the functions and mechanisms of unique radical SAM enzymes in methanogens, providing new insight into the biochemistry of these organisms, which are essential regulators of our climate. This work will supply the biochemical foundation for developing novel inhibitors against methanogenesis to decrease the methane released into our atmosphere as well as the foundation for generating liquid fuels from methane by harnessing the biochemistry of methanogenesis.Objective 1: Determine the functions and enzymatic mechanisms of methylthiotransferases in methanogens The methylthio group is found in a few select biological molecules, the most well-known of which is the amino acid methionine. The methylthio group imparts new chemical properties that result in specific bioactivities. In some cases, this group is added in two steps where sulfur is first incorporated to generate a thiol, and the resulting thiol is then methylated by traditional SAM-dependent nucleophilic substitution chemistry.26 In other cases, however, the site of the methylthio addition is unreactive and thus cannot undergo this chemistry. In these cases, the methylthio group is added by a radical SAM methylthiotransferase (MTTase).27 These enzymes are exemplified by MiaB and RimO, the enzymes catalyzing the synthesis of 2-methylthioadenosine in specific tRNAs and 3-methylthioaspartyl in the ribosomal S12 protein, respectively. The tRNA modification is involved in stabilizing the codon-anticodon interaction and thus is important for translational fidelity28 and the ribosomal S12 modification is proposed to be involved in translocation or decoding.29 MTTases first generate a methylthio group attached to a 4Fe-4S cluster and then add the methylthio group to a substrate radical.30 The reaction involves two 4Fe-4S clusters, one performs the radical SAM chemistry, and the other synthesizes the methylthio group. M. maripaludis contains three putative MTTases, MMP0047, MMP0412, and MMP1350, while Me. acetivorans has two, MA0242 and MA1153. None of these methanogenic MTTases have been functionally validated or enzymatically characterized. Based on sequence homology, MMP0412 and MA1153 are expected to be "e-MtaB", which catalyzes the conversion of N6-threonylcarbamoyladenosine to 2-methylthio-N6-threonylcarbamoyladenosine.31 MMP0047 and MA0242 share high homology to each other, however, they have completely unknown functions. Additionally, the function of MMP1350 is unknown. In a transposon mutagenesis study, MMP0412 was shown to be an essential gene, and both MMP0047 and MMP1350 were "likely essential".32 This indicates that these will be good potential future targets for inhibiting methanogenesis. Methanogens and related organisms called anaerobic methanotrophs contain a specialized coenzyme with a methylthio/thioether modification. Coenzyme F430 is the prosthetic group of methyl coenzyme M reductase (MCR), which catalyzes the final step of methanogenesis. The roles of the coenzyme F430 variants remains unclear, but an attractive possibility is that they tune MCR to perform methane oxidation instead of methane formation. Due to the unreactive nature of the site of modification, it is likely that the biosynthesis of these F430 variants uses an MTTase. We hypothesize that one of the MTTases with an unknown function in the methanogens catalyzes the biosynthesis of mercaptopropionate-F430. Since both M. maripaludis and Me. acetivorans contain mercaptopropionate-F430 (K. Allen, unpublished data) and since Me. acetivorans only contains one putative MTTase with an unknown function, the best candidate for mercaptopropionate-F430 biosynthesis is MA0242/MMP0047.Objective 2: Determine the enzymatic mechanism and the importance of the methylases in methanopterin biosynthesis. Methanopterinis a one-carbon (C1) carrier coenzyme that acts in three of the seven steps of hydrogenotrophic methanogenesis. MPT is structurally and functionally similar to folate, the canonical C1 carrier involved in a variety of essential biochemical processes. During the biosynthesis of MPT, two methyl groups are introduced at the C-7 and C-9 positions. The enzyme responsible for the addition of these methyl groups in the methanogen Methanocaldococcus jannaschii is MJ0619.25 However, the enzyme has not yet been studied in vitro and the substrate for the methylation reactions remains unconfirmed. Additionally, the methyl group donor for the reactions is not SAM as is the case with other radical SAM methyltransferases33 and, thus, the methyl group donor needs to be identified.Although M. maripaludis does not have a homolog to MJ0619, the Me. acetivorans genome encodes two proteins (MA114 and MA1486) with very high homology to MJ0619. At least one of these is likely catalyzing methylation reaction(s) in MPT biosynthesis in Me. acetivorans, and it is possible that each enzyme catalyzes single consecutive methylation reactions, instead of a single enzyme catalyzing both methylation reactions as is the case with MJ0619. The biochemical significance of the methyl groups on MPT has never been addressed in vivo, although theoretical biophysical and thermodynamic aspects have been discussed.34 Generating a methanogen strain that cannot synthesize the complete methylated MPT will provide insight into the importance of these methyl groups in allowing the coenzyme to bind to its partner enzymes and catalyze essential reactions in methanogenic metabolism.
Project Methods
Summary: Both in vitro and in vivo approaches will be used to determine the functions and mechanisms of selected radical SAM enzymes in methanogens. The in vitro approaches will involve cloning and expression of the his-tagged radical SAM enzyme of interest in E. coli. This will allow us to obtain large quantities of protein that can be purified and enzymatically characterized. Radical SAM enzymes contain at least one [4Fe-4S] cluster and require this cluster to be intact for folding and activity. In some cases this can become problematic if the cluster is not properly assembled. To overcome this, we can co-express radical SAM enzymes with Fe-S cluster assembly proteins. Another potential limitation is that the [4Fe-4S] cluster makes theses enzymes very sensitive to oxygen. Thus, generally all purification and enzymatic assays will take place in an anaerobic chamber. To understand the physiological importance of the enzymes of interest, we will carry out in vivo experiments involving the generation of deletion strains using established methods.37, 38 In some cases, the gene may be essential and so we will be unable to generate a deletion strain. As an alternative, we can instead use overexpression strains to address physiological function.Statistical analyses: The in vivo experiments will be performed in three independent replicates and also analyzed by liquid chromatography-mass spectrometry (LC-MS) in three separate runs. This will allow us to generate means and standard deviations for specific metabolite abundances that will then allow us to infer gene function. For in vitro assays, these will also be performed at least in triplicate for determining means and standard deviations for the various enzyme activities.Objective 1: Determine the functions and enzymatic mechanisms of methylthiotransferases in methanogensSummary: Based on their high sequence identity to e-MtaB, we hypothesize that the homologous enzymes, MMP0412 and MA1153, catalyze the synthesis of 2-methylthio-N6-threonylcarbamoyl-adenosine.31We also propose that MMP0047 and MA0242catalyze the thioether modification of mercaptopropionate-F430 (Fig. 6). The final radical SAM MTTase present in the genomes of our model methanogens is MMP1350, which does not have an associated homolog in Me. acetivorans. This enzyme likely catalyzes a methylthio group addition to an alternate tRNA substrate.Approach: In vitro. We have already cloned MA0242 and MMP1350 into pET15b plasmids for overexpression in E. coli and are in the process of cloning the other radical SAM MTTases for expression and purification from E. coli. For the methanogenic e-MtaB enzymes, we will adapt published procedures for the enzymatic activity determination of radical SAM MTTases.30 Similar methods will be employed for enzyme activity experiments to test whether MMP0047/MMP0242 catalyzes the synthesis of mercaptopropionate-F430, however, we will use the canonical F430purified from methanogen cells as a substrate. It is possible that MMP0047/MMP0242 only catalyze the initial addition of a sulfur containing intermediate to F430 and that other enzyme(s) are required for the complete synthesis of mercaptopropionate-F430. So, various potential sulfur-containing F430 intermediates will be assayed for by LC-MS. We have already demonstrated the application of sensitive LC-MS methods to analyze several F430-related compounds.39In vivo. Due to the likely essential nature of methanogenic MTTases based on published non-targeted transposon mutagenesis studies,32 generating deletions of these genes will likely not be feasible. Instead, the MTTases with unknown functions will be overexpressed in M. maripaludis and an increase in the hypothesized products of these enzymes (eg. mercaptopropionate-F430) will be analyzed by LC-MS.Objective 2: Investigate the enzymatic mechanisms and physiological significance of the methylases in methanopterin biosynthesis. Summary: MJ0619 from Met. jannaschii catalyzes methylation reactions in MPT biosynthesis, but the mechanism is distinct from other radical SAM methylases in that the methyl group donor is not SAM or methylcobalamin. We hypothesize that MJ0619. utilizes methylene-tetrahydrofolate/methylene-tetrahydromethanopterin as a methyl group donor, similar to what is observed with the methylation reaction catalyzed by thymidylate synthase. We also hypothesize that at least one of the MJ0619 homologs in Me. acetivorans catalyzes MPT methylation. Approach: In vitro. We have cloned MJ0619 into an E. coli overexpression plasmid30 and have purified small quantities of soluble enzyme. However, the majority of the enzyme is insoluble under normal expression conditions in E. coli. Therefore, we will adapt established procedures for improving solubility of radical SAM enzymes including co-expression with isc operon proteins, low temperature expression, and/or anaerobic/partially anaerobic expression conditions. The in vitro activity of MJ0619 will then be tested with various folate biosynthetic precursors and various potential methyl group donors. Once we identify the reaction conditions and co-substrates, including the methyl group donor, that supports the highest activity, we will perform isotopic labeling studies to support our hypothesis of a thymidylate synthase-like mechanism.In vivo. Since Met. jannaschii does not have a genetic system, the in vivo importance of MJ0619 cannot be addressed directly. Therefore, we will analyze the functions and importance of likely MPT methylases in vivo by generating a deletion of MJ0619 homologs in Me. acetivorans and analyzing the methylation status of MPT by LC-MS. Not only will this demonstrate whether the Me. acetivorans genes are involved in MPT methylation, it will also provide insight into the importance of the methyl groups on MPT and address whether they are they required for the function of the coenzyme.