Lactate metabolism in strictly anaerobic microorganisms with a soluble NAD+‐dependent l‐lactate dehydrogenase
Summary Lactate is a universal metabolite and energy source, yet the mode of lactate metabolism in many strictly anaerobic microorganisms is still enigmatic. This sparked us to investigate the biochemistry and bioenergetics of lactate metabolism in the model acetogenic bacterium Moorella thermoaceti...
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| Veröffentlicht in: | Environmental microbiology 2021-08, Vol.23 (8), p.4661-4672 |
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| creator | Rosenbaum, Florian P. Poehlein, Anja Egelkamp, Richard Daniel, Rolf Harder, Sönke Schlüter, Hartmut Schoelmerich, Marie Charlotte |
| description | Summary
Lactate is a universal metabolite and energy source, yet the mode of lactate metabolism in many strictly anaerobic microorganisms is still enigmatic. This sparked us to investigate the biochemistry and bioenergetics of lactate metabolism in the model acetogenic bacterium Moorella thermoacetica. Growth and metabolism were dependent on CO2 and the chemiosmotic gradient. We discovered a l‐lactate:NAD+ oxidoreductase (LDH) in cell‐free extracts, exhibiting an average specific activity of 362.8 ± 22.9 mU mg−1. The enzyme was reversible, most active at 65°C and pH 9, with Km values of 23.1 ± 3.7 mM for l‐lactate and 273.3 ± 39.1 μM for NAD+. In‐gel activity assays and mass spectrometric proteomics revealed that the ldh gene encoded the characterized LDH. Transcriptomic and genomic analyses showed that ldh expression was induced by lactate and there was a single nucleotide polymorphism near the predicted NAD+ binding site. Genes encoding central redox and energy metabolism complexes, such as, the energetic coupling site Ech2, menaquinone, and the electron bifurcating EtfABCX and MTHFR were also upregulated in cells grown on lactate. These findings ultimately lead to a redox‐balanced metabolic model that shows how growth on lactate can proceed in a microorganism that only has a conventional NAD+‐reducing LDH. |
| doi_str_mv | 10.1111/1462-2920.15657 |
| format | Article |
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Lactate is a universal metabolite and energy source, yet the mode of lactate metabolism in many strictly anaerobic microorganisms is still enigmatic. This sparked us to investigate the biochemistry and bioenergetics of lactate metabolism in the model acetogenic bacterium Moorella thermoacetica. Growth and metabolism were dependent on CO2 and the chemiosmotic gradient. We discovered a l‐lactate:NAD+ oxidoreductase (LDH) in cell‐free extracts, exhibiting an average specific activity of 362.8 ± 22.9 mU mg−1. The enzyme was reversible, most active at 65°C and pH 9, with Km values of 23.1 ± 3.7 mM for l‐lactate and 273.3 ± 39.1 μM for NAD+. In‐gel activity assays and mass spectrometric proteomics revealed that the ldh gene encoded the characterized LDH. Transcriptomic and genomic analyses showed that ldh expression was induced by lactate and there was a single nucleotide polymorphism near the predicted NAD+ binding site. Genes encoding central redox and energy metabolism complexes, such as, the energetic coupling site Ech2, menaquinone, and the electron bifurcating EtfABCX and MTHFR were also upregulated in cells grown on lactate. These findings ultimately lead to a redox‐balanced metabolic model that shows how growth on lactate can proceed in a microorganism that only has a conventional NAD+‐reducing LDH.</description><identifier>ISSN: 1462-2912</identifier><identifier>EISSN: 1462-2920</identifier><identifier>DOI: 10.1111/1462-2920.15657</identifier><language>eng</language><publisher>Hoboken, USA: John Wiley & Sons, Inc</publisher><subject>Anaerobic microorganisms ; Binding sites ; Bioenergetics ; Carbon dioxide ; Energy metabolism ; Energy sources ; Gels ; Gene expression ; Gene polymorphism ; Genomic analysis ; L-Lactate dehydrogenase ; Lactate ; Lactate dehydrogenase ; Lactic acid ; LDH gene ; Menaquinones ; Metabolism ; Metabolites ; Methylenetetrahydrofolate reductase ; Microorganisms ; NAD ; Nucleotides ; Oxidoreductase ; Oxidoreductases ; Oxidoreductions ; Polymorphism ; Proteomics ; Single-nucleotide polymorphism ; Spectrometry</subject><ispartof>Environmental microbiology, 2021-08, Vol.23 (8), p.4661-4672</ispartof><rights>2021 The Authors. published by Society for Applied Microbiology and John Wiley & Sons Ltd.</rights><rights>2021. This article is published under http://creativecommons.org/licenses/by-nc-nd/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3897-4a1ff67d3f5dd46246e33f0cd88af5724fcc58dac0a8ca56cc19c37c864b7e343</citedby><cites>FETCH-LOGICAL-c3897-4a1ff67d3f5dd46246e33f0cd88af5724fcc58dac0a8ca56cc19c37c864b7e343</cites><orcidid>0000-0002-8646-7925 ; 0000-0001-7679-0043 ; 0000-0002-2473-6202</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1111%2F1462-2920.15657$$EPDF$$P50$$Gwiley$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1111%2F1462-2920.15657$$EHTML$$P50$$Gwiley$$Hfree_for_read</linktohtml><link.rule.ids>314,776,780,1411,27903,27904,45553,45554</link.rule.ids></links><search><creatorcontrib>Rosenbaum, Florian P.</creatorcontrib><creatorcontrib>Poehlein, Anja</creatorcontrib><creatorcontrib>Egelkamp, Richard</creatorcontrib><creatorcontrib>Daniel, Rolf</creatorcontrib><creatorcontrib>Harder, Sönke</creatorcontrib><creatorcontrib>Schlüter, Hartmut</creatorcontrib><creatorcontrib>Schoelmerich, Marie Charlotte</creatorcontrib><title>Lactate metabolism in strictly anaerobic microorganisms with a soluble NAD+‐dependent l‐lactate dehydrogenase</title><title>Environmental microbiology</title><description>Summary
Lactate is a universal metabolite and energy source, yet the mode of lactate metabolism in many strictly anaerobic microorganisms is still enigmatic. This sparked us to investigate the biochemistry and bioenergetics of lactate metabolism in the model acetogenic bacterium Moorella thermoacetica. Growth and metabolism were dependent on CO2 and the chemiosmotic gradient. We discovered a l‐lactate:NAD+ oxidoreductase (LDH) in cell‐free extracts, exhibiting an average specific activity of 362.8 ± 22.9 mU mg−1. The enzyme was reversible, most active at 65°C and pH 9, with Km values of 23.1 ± 3.7 mM for l‐lactate and 273.3 ± 39.1 μM for NAD+. In‐gel activity assays and mass spectrometric proteomics revealed that the ldh gene encoded the characterized LDH. Transcriptomic and genomic analyses showed that ldh expression was induced by lactate and there was a single nucleotide polymorphism near the predicted NAD+ binding site. Genes encoding central redox and energy metabolism complexes, such as, the energetic coupling site Ech2, menaquinone, and the electron bifurcating EtfABCX and MTHFR were also upregulated in cells grown on lactate. These findings ultimately lead to a redox‐balanced metabolic model that shows how growth on lactate can proceed in a microorganism that only has a conventional NAD+‐reducing LDH.</description><subject>Anaerobic microorganisms</subject><subject>Binding sites</subject><subject>Bioenergetics</subject><subject>Carbon dioxide</subject><subject>Energy metabolism</subject><subject>Energy sources</subject><subject>Gels</subject><subject>Gene expression</subject><subject>Gene polymorphism</subject><subject>Genomic analysis</subject><subject>L-Lactate dehydrogenase</subject><subject>Lactate</subject><subject>Lactate dehydrogenase</subject><subject>Lactic acid</subject><subject>LDH gene</subject><subject>Menaquinones</subject><subject>Metabolism</subject><subject>Metabolites</subject><subject>Methylenetetrahydrofolate reductase</subject><subject>Microorganisms</subject><subject>NAD</subject><subject>Nucleotides</subject><subject>Oxidoreductase</subject><subject>Oxidoreductases</subject><subject>Oxidoreductions</subject><subject>Polymorphism</subject><subject>Proteomics</subject><subject>Single-nucleotide polymorphism</subject><subject>Spectrometry</subject><issn>1462-2912</issn><issn>1462-2920</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>24P</sourceid><sourceid>WIN</sourceid><recordid>eNqFkc9OAjEQxjdGExE9e23ixcQg2-223T0SRCVBvei5Ke0slHS30C4h3HwEn9EnsQjh4MW5zJ_8vsnkmyS5xuk9jtHHOct6WZnFljLKT5LOcXJ6rHF2nlyEsEhTzAlPO8lqIlUrW0A1tHLqrAk1Mg0KrTeqtVskGwneTY1CtVHeOT-TTWQC2ph2jiQKzq6nFtDr4OHu-_NLwxIaDU2LbOzsYbeG-VZ7N4NGBrhMzippA1wdcjf5eBy9D597k7en8XAw6SlSlLyXS1xVjGtSUa3j8TkDQqpU6aKQFeVZXilFCy1VKgslKVMKl4pwVbB8yoHkpJvc7vcuvVutIbSiNkGBtbIBtw4iozkreUFSHNGbP-jCrX0Tr4sUoxiXBWeR6u-p6EMIHiqx9KaWfitwKnYvEDuTxc5w8fuCqKB7xcZY2P6Hi9HLeK_7AWV4i64</recordid><startdate>202108</startdate><enddate>202108</enddate><creator>Rosenbaum, Florian P.</creator><creator>Poehlein, Anja</creator><creator>Egelkamp, Richard</creator><creator>Daniel, Rolf</creator><creator>Harder, Sönke</creator><creator>Schlüter, Hartmut</creator><creator>Schoelmerich, Marie Charlotte</creator><general>John Wiley & Sons, Inc</general><general>Wiley Subscription Services, Inc</general><scope>24P</scope><scope>WIN</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QH</scope><scope>7QL</scope><scope>7ST</scope><scope>7T7</scope><scope>7TN</scope><scope>7U9</scope><scope>7UA</scope><scope>8FD</scope><scope>C1K</scope><scope>F1W</scope><scope>FR3</scope><scope>H94</scope><scope>H95</scope><scope>H97</scope><scope>L.G</scope><scope>M7N</scope><scope>P64</scope><scope>SOI</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0002-8646-7925</orcidid><orcidid>https://orcid.org/0000-0001-7679-0043</orcidid><orcidid>https://orcid.org/0000-0002-2473-6202</orcidid></search><sort><creationdate>202108</creationdate><title>Lactate metabolism in strictly anaerobic microorganisms with a soluble NAD+‐dependent l‐lactate dehydrogenase</title><author>Rosenbaum, Florian P. ; 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Lactate is a universal metabolite and energy source, yet the mode of lactate metabolism in many strictly anaerobic microorganisms is still enigmatic. This sparked us to investigate the biochemistry and bioenergetics of lactate metabolism in the model acetogenic bacterium Moorella thermoacetica. Growth and metabolism were dependent on CO2 and the chemiosmotic gradient. We discovered a l‐lactate:NAD+ oxidoreductase (LDH) in cell‐free extracts, exhibiting an average specific activity of 362.8 ± 22.9 mU mg−1. The enzyme was reversible, most active at 65°C and pH 9, with Km values of 23.1 ± 3.7 mM for l‐lactate and 273.3 ± 39.1 μM for NAD+. In‐gel activity assays and mass spectrometric proteomics revealed that the ldh gene encoded the characterized LDH. Transcriptomic and genomic analyses showed that ldh expression was induced by lactate and there was a single nucleotide polymorphism near the predicted NAD+ binding site. Genes encoding central redox and energy metabolism complexes, such as, the energetic coupling site Ech2, menaquinone, and the electron bifurcating EtfABCX and MTHFR were also upregulated in cells grown on lactate. These findings ultimately lead to a redox‐balanced metabolic model that shows how growth on lactate can proceed in a microorganism that only has a conventional NAD+‐reducing LDH.</abstract><cop>Hoboken, USA</cop><pub>John Wiley & Sons, Inc</pub><doi>10.1111/1462-2920.15657</doi><tpages>12</tpages><orcidid>https://orcid.org/0000-0002-8646-7925</orcidid><orcidid>https://orcid.org/0000-0001-7679-0043</orcidid><orcidid>https://orcid.org/0000-0002-2473-6202</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Anaerobic microorganisms Binding sites Bioenergetics Carbon dioxide Energy metabolism Energy sources Gels Gene expression Gene polymorphism Genomic analysis L-Lactate dehydrogenase Lactate Lactate dehydrogenase Lactic acid LDH gene Menaquinones Metabolism Metabolites Methylenetetrahydrofolate reductase Microorganisms NAD Nucleotides Oxidoreductase Oxidoreductases Oxidoreductions Polymorphism Proteomics Single-nucleotide polymorphism Spectrometry |
| title | Lactate metabolism in strictly anaerobic microorganisms with a soluble NAD+‐dependent l‐lactate dehydrogenase |
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