Methanol-specific methyltransferase isozymes have large carbon kinetic isotope effects that impact methane isotopic...

The stable carbon and hydrogen isotopic composition of methane is widely used to determine its sources. Methanogenic growth on methanol generates methane with significantly lower ¹³C/¹²C 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 Methanosarcina acetivorans 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 (¹³ε_MTA) to be -66‰ and the hydrogen KIE of MTA (²ε_MTA) to be -56‰. Our results indicate that the ¹³ε_MTA 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 ¹³ε_MTA and ²ε_MTA 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 ¹³ε_MTA would be expressed in situ as well.IMPORTANCEMapping 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, Methanosarcina acetivorans. By utilizing M. acetivorans 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.