The stable hydrogen and carbon isotopic composition of methane is widely used to determine its sources. Methanogenic growth on methanol generates methane with significantly lower ¹³ C/ ¹² C ratios relative to other substrates, which is often used as a marker for this metabolism in environmental samples. The biochemical basis for the unusual isotope effect associated with methanol growth is currently unknown. Here, we grew Methanosarcina acetivorans on methanol and measured the change in the carbon and hydrogen stable isotopic compositions of the methane. We coupled 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 ( ¹³ ε _MTA ) as −65.5 ‰ and the hydrogen KIE of MTA ( ² ε _MTA ) as −56 ‰. Next, we show that the ¹³ ε _MTA contributes substantially to the large isotopic effect observed for methylotrophic methanogenesis on methanol. We also show that mutant strains that express only a single copy of the MTA complex (either MtaC1B1A1, MtaC2B2A1, or MtaC3B3A1) have ¹³ ε _MTA and ² ε _MTA that are indistinguishable from the wild-type strain. Finally, based on a thermodynamic analysis, we propose that methanol activation by MTA will remain rate-limiting, even at low environmental methanol concentrations, and the large ¹³ ε _MTA would be expressed in situ as well. Importance: Methane is a potent greenhouse gas, and distinguishing between its biological sources is vital for modeling global carbon cycles. Methylotrophic methanogenesis produces methane with a uniquely depleted carbon isotope signature. However, the biochemical mechanisms driving this fractionation have remained unclear. In this study, we identify the methanol-specific methyltransferase (MTA) complex as the primary driver of these large carbon isotope effects. By utilizing Methanosarcina acetivorans mutants, we demonstrate that these effects are consistent across different MTA isozymes. Our results suggest these signatures are intrinsic to the enzyme complex and persist at low substrate concentrations. These findings provide a critical biochemical foundation for using stable isotopes to track microbial methane production in diverse natural ecosystems.

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