Progress 01/01/15 to 12/31/18
Outputs
Target Audience:Our target audience is geochemists and biochemists interested in methanogenic reactions and energy production. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has trained a total of one PhD student and two undergraduates. How have the results been disseminated to communities of interest?Publications in scientific jounrals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We have made additional progress in establishing new metabolites and identifying the genes involved in their biosynthesis. This information is listed below. Discovery and identification a new thioquinoxalinol-containing redox Theanaerobicoxidation ofmethane (AOM) mitigates the flux of methane from marine sediments into the water column. AOM is performed byanaerobicmethanotrophic archaea (ANME) that reverse the methanogenesis pathway and partner bacteria that utilize the released reducing equivalents for sulfate reduction. We used high-resolution mass spectrometry to investigate small-molecule extracts from sediment-free thermophilic enrichment cultures of ANME-1 and sulfate-reducing bacteria. During the analysis, we discovered and characterized a novel thioquinoxalinol-containing redox molecule that contains both a redox-active quinoxaline heterocyclic ring and a thiol group. Additionally, the same structure was identified that contains a sulfate ester on the hydroxyl group, which likely makes the molecule more water-soluble. Hydrated versions of both structures were also observed as major compounds in the small-molecule extracts. On the basis of reactions of model compounds such as quinoxalin-6-ol, the hydrated version appears to be formed from the addition of water to the dehydropyrazine ring followed by an oxidation. We propose that these compounds, which represent completely new structures in biochemistry, could be involved in transporting electrons from ANME-1 to the sulfate-reducing bacteria. Our paper on this work has just been accepted in ACS Omega. Widespread occurrence of organisms producing amorphous carbon During our work on the analysis of metabolites in ANME cells as well as some methanogens we have recently found that they produce amorphous or black carbon. This is the first report of the production of amorphous carbon by any organism. Current data indicate that the carbon is produced from formate derived from formyl-tetrahydromethanopterin a compound found in these organisms. Its production may serve as an electron sink for the ANME organisms. At present we have no clear idea of how this form of carbon could be biochemically produced. Based on its chemical structure there appears to be no known biochemical mechanism for its production. Its identification in these organisms, which have a wide distribution in ocean sediments, is likely to have profound implications for carbon metabolism on earth. Our goal for the coming year will be to determine how it is formed. Metabolism of cysteine and homocysteine in methanogens. In an analysis of thiol-containing compounds in methanogenic archaea, we have identified isocysteine (3-amino-2-mercaptoproponic acid) and isohomocysteine (4-amino-2-mercaptobutanoic acid), which have never before been reported as natural products. The addition of labeled D/L-[3,3'-2H2]-cysteine to growing cultures of eitherMethanococcus maripaludisor its cysteine autotroph (DsepS) leads to the production of [3,3'-2H2]-isocysteine. Similarly, growingM. maripaludiswith [3,3',4,4'-4H2]-homocysteine leads to the production of the isohomocysteine isomer 4-amino-2-mercaptobutanoic acid. In both cases, each isomer contains the full complement of deuteriums from the respective precursor molecule. We propose that a single radical-dependent enzyme may be catalyzing the production of each of these molecules. The manuscript describing this work is currently being prepared.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
White, RH. Identification of an Enzyme Catalyzing the Conversion of Sulfoacetaldehyde to 2-Mercaptoethanesulfonic Acid in Methanogens. Biochemistry. 58(15):1958-1962. doi: 10.1021/acs.biochem.9b00176.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Portugal R, Shao N, Whitman WB, Allen KD, White RH. Identification and biosynthesis of 2-(1H-imidazol-5-yl) ethan-1-ol (histaminol) in methanogenic archaea. Microbiology. 165(4):455-462. doi: 10.1099/mic.0.000779.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:I conduct basic research in the identification of naturally occurring metabolites and their biosynthesis in methanogens. The target audiences are biochemists, microbiologist, and bioinformaticians. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?I currently have two undergraduate students working in the lab. How have the results been disseminated to communities of interest?As publications within scientific journals What do you plan to do during the next reporting period to accomplish the goals?Confirm the genes involved in the biosynthesis of F430-2 and histaminol, as a way to discern new engineering targets for altering methanogens.
Impacts
What was accomplished under these goals?
We have madeprogress in establishing new reactions and identifying the genes involved in their biosynthesis. This information will be useful for designing future engineering strategies to mitigate methane production in the environment.This information is listed below. 1. Widespread occurrence of histaminol in the methanogens and its biosynthesis Histaminol, an analog of histamine, is a relatively rare metabolite most commonly resulting from histidine metabolism. We found histaminol production and secretion into the culture broth by the methanogen Methanococcus maripaludis S2, as well as a host of other methanogens. This work is the first identification of this compound as a natural product produced in methanogens. Its biosynthesis from histidine was confirmed by the incorporation of 2H3-histidine into histaminol by growing cells of M. maripaludis S2. Possible functions of this molecule could be cell signaling as observed with histamine in eukaryotes or uptake of metal ions. Labeling studies with deuterated histidine show that the C-2, C'-2, and C'-4 deuteriums are incorporated into the histaminol. To explain this incorporation pattern, we propose that the enzyme is related to the SAM radical enzyme BlsE which catalyzes the non-oxidative decarboxylation of cytosylglucuronic acid (CGA) that occurs in the biosynthesis of blasticidin. Since we have found that ANMO-1 also contains histaminol we have good genomic evidence that MMP544 CKVKCNLC is a member of the MoaA/nifB/pqqE family of proteins (MJ0808 annonated as a pyruvate-formate lyase-activating enzyme (333 aa) CHICPRHC) is a likely the one. Other pathways from histidine to histaminol were eliminated through additional labeling studies. This MMP544 gene is proposed to be the enzyme involved. 2. Identification of a chiral sulfoxide in a modified F430 coenzyme in ANMO-1 During the analyses of the metabolites from an ANMO-1 coculture we have found a modified F430-2 we are designating as F430-2', where the methyl sulfide group at C-172 of F430-2 has been converted into a sulfoxide. Analysis of the different isomers of this F430-2' as well as that seen for F430-2 strongly indicate that the sulfoxide group is chiral. The occurrence of a chiral sulfoxide indicates that this functional group is enzymatically generated and is produced via an oxidation that proceeds in the absence of O2 or H2O2. We propose that this formation of the methyl sulfide group is catalyzed by radical SAM depend enzymes. 3. Identification of a method to confirm the presence of persulfides in biological samples. Here we have found a method for the analysis of persulfide in biological samples. The method rests on our observation that tris(2-carboxyethyl)phosphine hydrochloride (TCEP) reacts with persulfides at rt to produce TCEP-S (3,3',3''-(thioxophosphanetriyl)tripropionic acid) that can be identified and quantitated by LC-ESI-MS. The method is very specific and is more sensitive that the classical thiocyanate method. 4. Wide-spread occurrence of new metal binding monothiols produced in methanogens We have found the wide spread occurrence of a series of monothiol containing compound produced and secreted into their growth media by methanogens. Most of these molecules are new and have never been reported as a natural product. They can be grouped into four groups. The first group consists of the modified amino acids isocysteine and isohomocysteine; the second group consists of thiomalate, 2-mercaptoacetic acid (2-MA), 3-mercaptopropionic acid (3-MPA), 3-mercaptolactate, thiolactate, and 2-mercapto-3-hydroxypropionic acid (MHPA); and the third group consists 2-mercaptoacetic acid, or 3-MPA linked via a amide bond of the amino acids, glycine, alanine, serine, or threonine and consisted of 2-mercaptoacetylglycine, 2-mercaptoacetylalanine or 3-mercaptopropionylglycine, 3-mercaptopropionylalanine, 3 or 2-mercaptopropionylserine, 2-mercaptoacetylhomoserine, 2-mercaptoacetylthreonine, mercaptohydroxypropionic acid, 2-mercaptopropionyl-aspartate and (2-mercaptopropionyl-glutamate. The fourth consists of g-glutamylcysteine and two isomers of this. We propose that these compounds function as metal chelators to bind and transport metals into these cells.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2018
Citation:
Allen, K. D., and White, R. H. (2018) Identification of the Radical SAM Enzymes Involved in the Biosynthesis of Methanopterin and Coenzyme F420 in Methanogens, Methods Enzymol 606, 461-483
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Rebecca Portugal, Nana Shao, William B. Whitman, Kylie D. Allen, and Robert H. White Identification and biosynthesis of 2-(1H-imidazol-5-yl)ethan-1-ol (histaminol) in methanogenic archaea
- Type:
Journal Articles
Status:
Submitted
Year Published:
2018
Citation:
Robert H. White Identification of a Coenzyme M Synthase: An Enzyme Catalyzing the Conversion of Sulfoacetaldehyde to 2-Mercaptoethane-sulfonic acid (HSCoM) in Methanogens
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Biochemists, biologists, graduate and undergraduate students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We are training 2 undergradrate students to do original biochemical research. How have the results been disseminated to communities of interest?Publication and conference presentations. What do you plan to do during the next reporting period to accomplish the goals?We will finalize several (up to 12) manuscripts and submit to scientific journals.
Impacts
What was accomplished under these goals?
I have identified 1-mercaptoethanesulfonic acid (1-MES), an analog of 2-mercaptoethanesulfonic acid (coenzyme M, HSCoM) in methanogens. 1-MES and HSCoM were both present in the growth media of eight different methanogens at concentrations ranging from ~1-100 mM. In an effort to determine a chemical origin of 1-MES several plausible chemical routes were examined each assuming that HSCoM was the precursor. In all examined routes, no 1-MES was formed. However, 1-MES was formed when a solution of vinylsulfonic acid and sulfide was exposed to UV light. Based on these results I concluded 1-MES is formed enzymatically. Weconfirmed this by growing a culture of Methanococcus maripaludis S2 in the presence of [1,1',2,2'-2H4]-HSCoM and measuring the incorporation of deuterium into 1-MES. 1-MES incorporated three of the four deuteriums from the fed HSCoM. This result is consistent with the abstraction of a C-2 deuterium of the HSCoM, likely by a 5'-dAdoCH2· radical, followed by a radical rearrangement in which the sulfonic acid moves to the C-1 position, followed by abstraction of a H· likely from 5'-dAdoCH2D. At present, the reason for the production of 1-MES is not clear. This is the first report of the occurrence of 1-MES in Nature. I propose that 1-MES along with the different modified F430 coenzymes identified in the methanogens are used to allow for the anaerobic oxidation of methane (AOM) by the methanogens. We are currently workingto determine the enzyme involved in catalyzing this reaction since it likely is very important in determining the amount of methane in the earths atmosphere.
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Our work is of interest to both biochemists and microbiologists, and will impact knowledge of metabolic pathways. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Our work supplies avenues for the training and professional development of graduate students. How have the results been disseminated to communities of interest?Publication in scientific journals. What do you plan to do during the next reporting period to accomplish the goals?We need to publish at least four manuscripts on our current work and continuework on establishing the enzymology of our newly identified enzymes.
Impacts
What was accomplished under these goals?
1. We have established most of the pathway for the recovery of SAM radical enzymes in the methanogens. This has involved the identification of 5 new enzymes involved in this pathway. 2. We have estblished the pathway for the biosynthesis of 3-mercaptopropionic acid in the methanogens and have proposed a reason for the production of the molecule in the methanogens. 3.We have estblished that PEP synthetase is responsible of the detoxification of the sulfide in the methanogens. 4. We have established that enzyme responsible for the biosynthesis of F430-5 in the methanogens.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Miller, D.V., Brown, A., Xu, H., Bevan, D., and White RH. (2016) Purine salvage in Methanocaldococcus jannaschii: Elucidating the role of the conserved cysteine in an adenine deaminase, Proteins: Structure, Function, and Bioinformatics 84, 828-840.
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Progress 01/01/15 to 09/30/15
Outputs
Target Audience:Our target audience will be scientists in the following areas: Microbiologists, biochemists, enzymologists, bioinformaticists, and metabolic engineering. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training of a graduate student and undergraduate students in methodology and evaluation of literature relevant to this problem. How have the results been disseminated to communities of interest?Scientific publications. What do you plan to do during the next reporting period to accomplish the goals?Establish the chemical structures of presently unknown untargeted water-soluble metabolites in selected methanogens. This will involve the analysis of current LC-MS data to determine possible structures followed by confirmation of these structures when knowns are available. Establish the pathway for the biosynthesis of these compounds. This will involve defining the pathways by determining possible precursors.
Impacts
What was accomplished under these goals?
Our lab has made significant advances in establishing new metabolism in the methanogen Methanocaldococcus jannaschii. Many of these findings have implications in developing our understanding on the origin of life. First we have established that homocysteine is biosynthesized from aspartate semialdehyde and hydrogen sulfide in the methanogens. This work has identified a completely new biosynthetic route to homocysteine that is the precursor to methionine. We have also discovered and identified multiple modified F430 coenzymes in methanogens and anaerobic methanotrophic archaea suggesting possible novel roles for F430 in Nature. Understanding the involvement of these new coenzymes could completely alter our current understanding of the metabolism of the methanogens. We have begun to explore the biosynthesis of one of these modified cofactors, F430-3, that contains the covalent addition of 3-mercaptopropionic acid. We have found that the added portion of F430-3 is derived from 3-mercaptopropionic acid which in turn derived from D-malate semialdehyde produced from 2-hydroxy-4-mercaptobutyric acid all of which are present in M. jannaschii. All of the genes except one have been elucidated for the biosynthesis methanofuran, an essentialcoenzymein methane formation. In our search to establish a new methionine salvage pathway in the methanogens we have identified and characterized methylthioinosine phosphorylase and methylthioribose-1-P isomerase. These were found to be generalist enzymes likely tobe involved in other aspects of the metabolism of this organism. Finally we have determined a new pathway for the biosynthesis of beta-alanine, a component of the coenzyme A M. jannaschii.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Allen, K. D., Wegener, G., and White, R. H. (2014) Discovery of multiple modified F430 coenzymes in methanogens and anaerobic methanotrophic archaea suggests possible new roles for F430 in Nature, Appl. Environ. Microbiol. 80, 6403-6412.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Wang, Y., Jones, M. K., Xu, H., Ray, W. K., and White, R. H. (2015) Mechanism of the Enzymatic Synthesis of 4-(Hydroxymethyl)-2- furancarboxaldehyde-phosphate (4-HFC-P) from Glyceraldehyde-3-phosphate Catalyzed by 4-HFC-P Synthase, Biochemistry 54, 2997-3008.
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