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Methanogenic growth on methanol generates methane with significantly lower\n                      <jats:sup>13<\/jats:sup>\n                      C\/\n                      <jats:sup>12<\/jats:sup>\n                      C ratios relative to other substrates, which is used as a marker for this metabolism in environmental samples. The biochemical basis for the large carbon isotope effect associated with methanol growth is currently unknown. Here, we measure the change in the carbon and hydrogen stable isotopic compositions of the methane produced by\n                      <jats:italic toggle=\"yes\">Methanosarcina acetivorans<\/jats:italic>\n                      during growth on methanol. We couple these results with an inverse modeling approach to calculate the kinetic isotopic effects (KIEs) of the rate-limiting step, catalyzed by the methanol-specific methyltransferase complex (MTA). Through this process, we estimate the carbon KIE of MTA (\n                      <jats:sup>13<\/jats:sup>\n                      <jats:italic toggle=\"yes\">\u03b5<\/jats:italic>\n                      <jats:sub>MTA<\/jats:sub>\n                      ) to be \u221266\u2030 and the hydrogen KIE of MTA (\n                      <jats:sup>2<\/jats:sup>\n                      <jats:italic toggle=\"yes\">\u03b5<\/jats:italic>\n                      <jats:sub>MTA<\/jats:sub>\n                      ) to be \u221256\u2030. Our results indicate that the\n                      <jats:sup>13<\/jats:sup>\n                      \u03b5\n                      <jats:sub>MTA<\/jats:sub>\n                      contributes substantially to the large carbon isotope effect observed on methanol. We also show that mutant strains that express only a single copy of the MTA complex (MtaC1B1A1, MtaC2B2A1, or MtaC3B3A1) have\n                      <jats:sup>13<\/jats:sup>\n                      <jats:italic toggle=\"yes\">\u03b5<\/jats:italic>\n                      <jats:sub>MTA<\/jats:sub>\n                      and\n                      <jats:sup>2<\/jats:sup>\n                      <jats:italic toggle=\"yes\">\u03b5<\/jats:italic>\n                      <jats:sub>MTA<\/jats:sub>\n                      that are indistinguishable from the wild-type strain. Finally, we propose that methanol activation by MTA will remain rate-limiting, even at low environmental methanol concentrations, and the large\n                      <jats:sup>13<\/jats:sup>\n                      <jats:italic toggle=\"yes\">\u03b5<\/jats:italic>\n                      <jats:sub>MTA<\/jats:sub>\n                      would be expressed\n                      <jats:italic toggle=\"yes\">in situ<\/jats:italic>\n                      as well.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Mapping the biological sources of methane is vital for accurately modeling global emissions of this potent greenhouse gas. Methanogenic growth on methanol produces methane with a uniquely depleted carbon isotope signature, which is commonly used as an environmental indicator of this metabolism. The biochemical mechanisms driving this isotopic signature have remained unclear. In this study, we identify the methanol-specific methyltransferase (MTA) complex as the primary driver of these large carbon isotope effects in the model methanogen,\n                        <jats:italic toggle=\"yes\">Methanosarcina acetivorans<\/jats:italic>\n                        . By utilizing\n                        <jats:italic toggle=\"yes\">M. acetivorans<\/jats:italic>\n                        mutants that express a single MTA complex, we demonstrate that these effects are consistent across MTA isozymes that are expressed during different growth phases. Our results suggest these isotopic signatures are intrinsic to the enzyme complex and persist in natural environments. These findings provide a critical biochemical foundation for using stable isotopes to track methane production from methanol in diverse natural ecosystems.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/aem.00664-26","type":"journal-article","created":{"date-parts":[[2026,7,21]],"date-time":"2026-07-21T13:00:26Z","timestamp":1784638826000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Methanol-specific methyltransferase isozymes have large carbon kinetic isotope effects that impact methane isotopic signatures"],"prefix":"10.1128","volume":"92","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1193-1735","authenticated-orcid":true,"given":"Jonathan","family":"Gropp","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01an7q238","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Earth and Planetary 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